Delivery, use and therapeutic applications of the CRISPR-Cas systems and compositions for genome editing

ABSTRACT

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a Continuation-in-Part of International ApplicationNumber PCT/US14/70127 filed on Dec. 12, 2014, which published as PCTPublication No. WO2015/089462 on Jun. 18, 2015. This application claimspriority to U.S. provisional patent applications: 61/915,176;61/915,192; 61/915,215; 61/915,107, 61/915,145; 61/915,148; and61/915,153 each filed Dec. 12, 2013.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. MH100706awarded by the National Institutes of Health. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy created, Jun. 9, 2016, isnamed 47627.00.2091_SL.txt is 57,990 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to the delivery, engineering,optimization and therapeutic applications of systems, methods, andcompositions used for the control of gene expression involving sequencetargeting, such as genome perturbation or gene-editing, that relate toClustered Regularly Interspaced Short Palindromic Repeats (CRISPR) andcomponents thereof. In particular, the present invention relates to invitro, ex vivo and/or in vivo systems, methods, and compositions fordelivery of the CRISPR-Cas system to achieve therapeutic benefits viagenome editing in animals, including mammals.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers (ZFN),transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome engineering technologies that are affordable, easyto set up, scalable, and amenable to targeting multiple positions withinthe eukaryotic genome.

SUMMARY OF THE INVENTION

Despite valid therapeutic hypotheses and strong efforts in drugdevelopment, there have only been a limited number of successes usingsmall molecules to treat diseases with strong genetic contributions.Thus, there exists a pressing need for alternative and robust systemsfor therapeutic strategies that are able to modify nucleic acids withindisease-affected cells and tissues. Adding the CRISPR-Cas system to therepertoire of therapeutic genome engineering methods significantlysimplifies the methodology and accelerates the ability to catalog andmap genetic factors associated with a diverse range of biologicalfunctions and diseases, develop animal models for genetic diseases, anddevelop safe, effective therapeutic alternatives. To utilize theCRISPR-Cas system effectively for genome editing without deleteriouseffects, it is critical to understand aspects of engineering,optimization and cell-type/tissue/organ specific delivery of thesegenome engineering tools, which are aspects of the claimed invention.Aspects of this invention address this need and provide relatedadvantages.

An exemplary CRISPR complex may comprise a CRISPR enzyme (e.g., Cas9)complexed with a guide sequence hybridized to a target sequence withinthe target polynucleotide. The guide sequence is linked to a tracr matesequence, which in turn hybridizes to a tracr sequence. Applicants haveoptimized components of the CRISPR-Cas genome engineering system,including using SaCas9 from Staphylococcus aureus. Various deliverymeans may be employed for delivering components of the CRISPR-Cas systemto cells, tissues and organs, ex vivo and/or in vivo. Applicants haveeffectively packaged CRISPR-Cas system components (e.g., comprisingSaCas9) into a viral delivery vector, e.g., AAV, and have demonstratedthat it can be used to modify endogenous genome sequence in mammaliancells in vivo. A key feature of Applicants' present invention it that iteffectively addresses the challenges of low efficiency of in vivodelivery (of therapeutic components) and low efficiency of homologydirected repair (HDR) and in particular challenges associated withco-delivery are solved by the small Cas9, SaCas9 from Staphylococcusaureus, which can be readily packaged into a single Adeno-associatedvirus (AAV) vector to express both the Cas9 protein and itscorresponding sgRNA(s). Further, importantly, Applicants have shown thatintroduction of small SaCas9, has reduced the number of viral vectorsrequired to perform HDR from 3 vectors to 2 vectors. In aspects of theinvention particles may be used for delivery of one or more componentsof the CRISPR-Cas system. And the number of particles to be contactedwith can be one or two. In one aspect, the invention provides methodsfor using one or more elements of a CRISPR-Cas system. The CRISPRcomplex of the invention provides an effective means for modifying atarget polynucleotide in a genomic locus, wherein the genomic locus isassociated with a mutation, including mutations associated with anaberrant protein expression or with a disease condition or state. TheCRISPR complex of the invention has a wide variety of utilitiesincluding modifying (e.g., deleting, inserting, translocating,inactivating, activating) a target polynucleotide within a genomiclocus, including within a coding, non-coding or regulatory element ofsuch a target locus. As such the CRISPR complex of the invention has abroad spectrum of applications in, e.g., gene or genome editing, genetherapy, drug discovery, drug screening, disease diagnosis, andprognosis. Aspects of the invention relate to Cas9 enzymes havingimproved targeting specificity in a CRISPR-Cas9 system having guide RNAshaving optimal activity, smaller in length than wild-type Cas9 enzymesand nucleic acid molecules coding therefor, and chimeric Cas9 enzymes,as well as methods of improving the target specificity of a Cas9 enzymeor of designing a CRISPR-Cas9 system comprising designing or preparingguide RNAs having optimal activity and/or selecting or preparing a Cas9enzyme having a smaller size or length than wild-type Cas9 wherebypackaging a nucleic acid coding therefor into a delivery vector is moreadvanced as there is less coding therefor in the delivery vector thanfor wild-type Cas9, and/or generating chimeric Cas9 enzymes. Alsoprovided are uses of the present sequences, vectors, enzymes or systems,in medicine. Also provided are uses of the same in gene or genomeediting.

In the invention, the Cas enzyme can be wildtype Cas9 including anynaturally-occurring bacterial Cas9. Cas9 orthologs typically share thegeneral organization of 3-4 RuvC domains and a HNH domain. The 5′ mostRuvC domain cleaves the non-complementary strand, and the HNH domaincleaves the complementary strand. All notations are in reference to theguide sequence. The catalytic residue in the 5′ RuvC domain isidentified through homology comparison of the Cas9 of interest withother Cas9 orthologs (from S. pyogenes type II CRISPR locus, S.thermophilus CRISPR locus 1, S. thermophilus CRISPR locus 3, andFranciscilla novicida type II CRISPR locus), and the conserved Aspresidue (D10) is mutated to alanine to convert Cas9 into acomplementary-strand nicking enzyme. Similarly, the conserved His andAsn residues in the HNH domains are mutated to Alanine to convert Cas9into a non-complementary-strand nicking enzyme. In some embodiments,both sets of mutations may be made, to convert Cas9 into a non-cuttingenzyme. Accordingly, the Cas enzyme can be wildtype Cas9 including anynaturally-occurring bacterial Cas9. The CRISPR, Cas or Cas9 enzyme canbe codon optimized for human cells, including specific types of humancells, or a modified version, including any chimeras, mutants, homologsor orthologs. In an additional aspect of the invention, a Cas9 enzymemay comprise one or more mutations and may be used as a generic DNAbinding protein with or without fusion to a functional domain. Themutations may be artificially introduced mutations or gain- orloss-of-function mutations. The mutations may include but are notlimited to mutations in one of the catalytic domains (D10 and H840) inthe RuvC and HNH catalytic domains, respectively. Further mutations havebeen characterized. In one aspect of the invention, the transcriptionalactivation domain may be VP64. In other aspects of the invention, thetranscriptional repressor domain may be KRAB or SID4X. Other aspects ofthe invention relate to the mutated Cas 9 enzyme being fused to domainswhich include but are not limited to a transcriptional activator,repressor, a recombinase, a transposase, a histone remodeler, ademethylase, a DNA methyltransferase, a cryptochrome, a lightinducible/controllable domain or a chemically inducible/controllabledomain. The invention can involve sgRNAs or tracrRNAs or guide orchimeric guide sequences that allow for enhancing performance of theseRNAs in cells. The CRISPR enzyme can be a type I or III CRISPR enzyme,preferably a type II CRISPR enzyme. This type II CRISPR enzyme may beany Cas enzyme. A preferred Cas enzyme may be identified as Cas9 as thiscan refer to the general class of enzymes that share homology to thebiggest nuclease with multiple nuclease domains from the type II CRISPRsystem. Most preferably, the Cas9 enzyme is from, or is derived from,spCas9 or saCas9. By derived, Applicants mean that the derived enzyme islargely based, in the sense of having a high degree of sequence homologywith, a wildtype enzyme, but that it has been mutated (modified) in someway as described herein.

It will be appreciated that the terms Cas and CRISPR enzyme aregenerally used herein interchangeably, unless otherwise apparent. Asmentioned above, many of the residue numberings used herein refer to theCas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.However, it will be appreciated that this invention includes many moreCas9s from other species of microbes, such as SpCas9, SaCas9, St1Cas9and so forth. Further examples are provided herein. The skilled personwill be able to determine appropriate corresponding residues in Cas9enzymes other than SpCas9 by comparison of the relevant amino acidsequences. Thus, where a specific amino acid replacement is referred tousing the SpCas9 numbering, then, unless the context makes it apparentthis is not intended to refer to other Cas9 enzymes, the disclosure isintended to encompass corresponding modifications in other Cas9 enzymes.An example of a codon optimized sequence, in this instance optimized forhumans (i.e. being optimized for expression in humans) is providedherein, see the SaCas9 human codon optimized sequence. Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species is known. The inventioncomprehends methods wherein the Cas9 is a chimeric Cas9 proteins. Thesemethods may comprise N-terminal fragment(s) of one Cas9 homolog withC-terminal fragment(s) of one or more other or another Cas9 homolog. Itwill be appreciated that in the present methods, where the organism isan animal, the modification may occur ex vivo or in vitro, for instancein a cell culture and in some instances not in vivo. In otherembodiments, it may occur in vivo. The invention comprehends in someembodiments a composition of the invention or a CRISPR enzyme thereof(including or alternatively mRNA encoding the CRISPR enzyme), whereinthe target sequence is flanked at its 3′ end by a PAM (protospaceradjacent motif) sequence comprising 5′-motif, especially where the Cas9is (or is derived from) S. pyogenes or S. aureus Cas9. For example, asuitable PAM is 5′-NRG or 5′-NNGRR (where N is any Nucleotide) forSpCas9 or SaCas9 enzymes (or derived enzymes). It will be appreciatedthat SpCas9 or SaCas9 are those from or derived from S. pyogenes or S.aureus Cas9.

In one aspect, the invention provides a method of modifying an organismor a non-human organism by manipulation of a target sequence in agenomic locus of interest, wherein the genomic locus is associated witha mutation associated with an aberrant protein expression or with adisease condition or state comprising:

delivering a non-naturally occurring or engineered compositioncomprising:

-   -   A) I. a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide        sequence, wherein the polynucleotide sequence comprises:        -   (a) a guide sequence capable of hybridizing to a target            sequence in a eukaryotic cell,        -   (b) a tracr mate sequence, and        -   (c) a tracr sequence, and    -   II. a polynucleotide sequence encoding a CRISPR enzyme        comprising at least one or more nuclear localization sequences,        wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the CRISPR enzyme complexed        with (1) the guide sequence that is hybridized to the target        sequence, and (2) the tracr mate sequence that is hybridized to        the tracr sequence and the polynucleotide sequence encoding a        CRISPR enzyme is DNA or RNA; and

the method may optionally include also delivering a HDR template, e.g.,via a viral delivery vector or a particle, the HDR template wherein theHDR template provides expression of a normal or less aberrant form ofthe protein; wherein “normal” is as to wild type, and “aberrant” can bea protein expression that gives rise to a condition or disease state;and

optionally the method may include isolating or obtaining cellsexpressing said aberrant protein from the organism or non-humanorganism, optionally expanding the cell population, performingcontacting of the viral vector or particle(s) with said cells to obtaina modified cell population, optionally expanding the population ofmodified cells, and optionally administering modified cells to theorganism or non-human organism.

In one aspect, the invention provides a method of modifying an organismor a non-human organism by manipulation of a target sequence in agenomic locus of interest, wherein the genomic locus of interest isassociated with a mutation associated with an aberrant proteinexpression or with a disease condition or state, comprising: contactinga cell with a viral vector or particle containing, a non-naturallyoccurring or engineered composition comprising: I. (a) a guide sequencecapable of hybridizing to a target sequence in a HSC, and (b) at leastone or more tracr mate sequences, II. a CRISPR enzyme optionally havingone or more NLSs, and III. a polynucleotide sequence comprising a tracrsequence, wherein the tracr mate sequence hybridizes to the tracrsequence and the guide sequence directs sequence-specific binding of aCRISPR complex to the target sequence, and wherein the CRISPR complexcomprises the CRISPR enzyme complexed with (1) the guide sequence thatis hybridized to the target sequence, and (2) the tracr mate sequencethat is hybridized to the tracr sequence; and

the method may optionally include also delivering a HDR template, e.g.,via a viral delivery vector or a particle, the HDR template wherein theHDR template provides expression of a normal or less aberrant form ofthe protein; wherein “normal” is as to wild type, and “aberrant” can bea protein expression that gives rise to a condition or disease state;and

optionally the method may include isolating or obtaining cellsexpressing said aberrant protein from the organism or non-humanorganism, optionally expanding the cell population, performingcontacting of the viral vector or particle(s) with said cells to obtaina modified cell population, optionally expanding the population ofmodified cells, and optionally administering modified cells to theorganism or non-human organism.

The delivery can be of one or more polynucleotides encoding any one ormore or all of the CRISPR-complex, advantageously linked to one or moreregulatory elements for in vivo expression, e.g. via particle(s),containing a vector containing the polynucleotide(s) operably linked tothe regulatory element(s). Any or all of the polynucleotide sequenceencoding a CRISPR enzyme, guide sequence, tracr mate sequence or tracrsequence, may be RNA. It will be appreciated that where reference ismade to a polynucleotide, which is RNA and is said to ‘comprise’ afeature such a tracr mate sequence, the RNA sequence includes thefeature. Where the polynucleotide is DNA and is said to comprise afeature such a tracr mate sequence, the DNA sequence is or can betranscribed into the RNA including the feature at issue. Where thefeature is a protein, such as the CRISPR enzyme, the DNA or RNA sequencereferred to is, or can be, translated (and in the case of DNAtranscribed first).

In certain embodiments the invention provides a method of modifying anorganism, e.g., mammal including human or a non-human mammal or organismby manipulation of a target sequence in a genomic locus of intereste.g., wherein the genomic locus of interest is associated with amutation associated with an aberrant protein expression or with adisease condition or state, comprising delivering, e.g., via contactingof a non-naturally occurring or engineered composition with a cell orcell population, wherein the composition comprises one or more deliveryvectors or particles comprising viral, plasmid or nucleic acid moleculevector(s) (e.g. RNA) operably encoding a composition for expressionthereof, wherein the composition comprises: (A) I. a first regulatoryelement operably linked to a CRISPR-Cas system chimeric RNA (chiRNA)polynucleotide sequence, wherein the polynucleotide sequence comprises(a) a guide sequence capable of hybridizing to a target sequence in aeukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence,and II. a second regulatory element operably linked to an enzyme-codingsequence encoding a CRISPR enzyme comprising at least one or morenuclear localization sequences (or optionally at least one or morenuclear localization sequences as some embodiments can involve no NLS),wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, whereincomponents I and II are located on the same or different vectors of thesystem, wherein when transcribed, the tracr mate sequence hybridizes tothe tracr sequence and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, and wherein theCRISPR complex comprises the CRISPR enzyme complexed with (1) the guidesequence that is hybridized to the target sequence, and (2) the tracrmate sequence that is hybridized to the tracr sequence, or (B) anon-naturally occurring or engineered composition comprising a vectorsystem comprising one or more vectors comprising I. a first regulatoryelement operably linked to (a) a guide sequence capable of hybridizingto a target sequence in a eukaryotic cell, and (b) at least one or moretracr mate sequences, II. a second regulatory element operably linked toan enzyme-coding sequence encoding a CRISPR enzyme, and III. a thirdregulatory element operably linked to a tracr sequence, whereincomponents I, II and III are located on the same or different vectors ofthe system, wherein when transcribed, the tracr mate sequence hybridizesto the tracr sequence and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, and wherein theCRISPR complex comprises the CRISPR enzyme complexed with (1) the guidesequence that is hybridized to the target sequence, and (2) the tracrmate sequence that is hybridized to the tracr sequence; the method mayoptionally include also delivering a HDR template, e.g., via thedelivery vectors or particle contacting the cell or cell population orcontacting the cell cell or cell population with another delivery vectoror particle containing, the HDR template wherein the HDR templateprovides expression of a normal or less aberrant form of the protein;wherein “normal” is as to wild type, and “aberrant” can be a proteinexpression that gives rise to a condition or disease state; andoptionally the method may include isolating or obtaining cellsexpressing said aberrant proteins from the organism or non-humanorganism, optionally expanding said cell population, performingcontacting of the delivery vector or particle(s) with said cellsexpressing said aberrant proteins to obtain a modified cell population,optionally expanding the population of modified cells and optionallyadministering modified cells to the organism or non-human organism. Insome embodiments, components I, II and III are located on the samevector. In other embodiments, components I and II are located on thesame vector, while component III is located on another vector. In otherembodiments, components I and III are located on the same vector, whilecomponent II is located on another vector. In other embodiments,components II and III are located on the same vector, while component Iis located on another vector. In other embodiments, each of componentsI, II and III is located on different vectors. The invention alsoprovides a viral or plasmid vector system as described herein.

By manipulation of a target sequence, Applicants also mean theepigenetic manipulation of a target sequence. This may be of thechromatin state of a target sequence, such as by modification of themethylation state of the target sequence (i.e. addition or removal ofmethylation or methylation patterns or CpG islands), histonemodification, increasing or reducing accessibility to the targetsequence, or by promoting 3D folding. It will be appreciated that wherereference is made to a method of modifying an organism or mammalincluding human or a non-human mammal or organism by manipulation of atarget sequence in a genomic locus of interest, this may apply to theorganism (or mammal) as a whole or just a single cell or population ofcells from that organism (if the organism is multicellular). In the caseof humans, for instance, Applicants envisage, inter alia, a single cellor a population of cells and these may preferably be modified ex vivoand then re-introduced. In this case, a biopsy or other tissue orbiological fluid sample may be necessary. Stem cells are alsoparticularly preferred in this regard. But, of course, in vivoembodiments are also envisaged. And the invention is especiallyadvantageous as to ocular cells, retinal cells, vascular cells,epithelial cells, endothelial cells, and cochlear cells.

The invention in some embodiments comprehends a method of modifying anorganism or a non-human organism by manipulation of a first and a secondtarget sequence on opposite strands of a DNA duplex in a genomic locusof interest in a cell or cell population e.g., wherein the genomic locusof interest is associated with a mutation associated with an aberrantprotein expression or with a disease condition or state, comprisingdelivering, e.g., by contacting the cell or cell population with adelivery vector, e.g., viral vectors or particles comprising anon-naturally occurring or engineered composition comprising:

-   -   I. a first CRISPR-Cas system chimeric RNA (chiRNA)        polynucleotide sequence, wherein the first polynucleotide        sequence comprises:        -   (a) a first guide sequence capable of hybridizing to the            first target sequence,        -   (b) a first tracr mate sequence, and        -   (c) a first tracr sequence,    -   II. a second CRISPR-Cas system chiRNA polynucleotide sequence,        wherein the second polynucleotide sequence comprises:        -   (a) a second guide sequence capable of hybridizing to the            second target sequence,        -   (b) a second tracr mate sequence, and        -   (c) a second tracr sequence, and    -   III. a polynucleotide sequence encoding a CRISPR enzyme        comprising at least one or more nuclear localization sequences        and comprising one or more mutations, wherein (a), (b) and (c)        are arranged in a 5′ to 3′ orientation; or    -   IV. expression product(s) of one or more of I. to III., e.g.,        the the first and the second tracr mate sequence, the CRISPR        enzyme;

wherein when transcribed, the first and the second tracr mate sequencehybridize to the first and second tracr sequence respectively and thefirst and the second guide sequence directs sequence-specific binding ofa first and a second CRISPR complex to the first and second targetsequences respectively, wherein the first CRISPR complex comprises theCRISPR enzyme complexed with (1) the first guide sequence that ishybridized to the first target sequence, and (2) the first tracr matesequence that is hybridized to the first tracr sequence, wherein thesecond CRISPR complex comprises the CRISPR enzyme complexed with (1) thesecond guide sequence that is hybridized to the second target sequence,and (2) the second tracr mate sequence that is hybridized to the secondtracr sequence, wherein the polynucleotide sequence encoding a CRISPRenzyme is DNA or RNA, and wherein the first guide sequence directscleavage of one strand of the DNA duplex near the first target sequenceand the second guide sequence directs cleavage of the other strand nearthe second target sequence inducing a double strand break, therebymodifying the organism or the non-human organism; and the method mayoptionally include also delivering a HDR template, e.g., via thedelivery vector contacting the cell or cell population containing orcontacting the cell or cell population with another delivery vectorcontaining, the HDR template wherein the HDR template providesexpression of a normal or less aberrant form of the protein; wherein“normal” is as to wild type, and “aberrant” can be a protein expressionthat gives rise to a condition or disease state; and optionally themethod may include isolating or obtaining a cell or cell population fromthe organism or non-human organism, optionally expanding the cellpopulation, performing contacting of the delivery vector or particle(s)with the cell or cell population to obtain a modified cell population,optionally expanding the population of modified cells A method ofmodeling a disease associated with a genomic locus in a eukaryoticorganism or a non-human organism comprising manipulation of a targetsequence within a coding, non-coding or regulatory element of saidgenomic locus comprising delivering a non-naturally occurring orengineered composition comprising a viral vector system comprising oneor more viral vectors operably encoding a composition for expressionthereof, wherein the composition comprises:

(A) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising

I. a first regulatory element operably linked to a CRISPR-Cas system RNApolynucleotide sequence, wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to the target sequence,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a second regulatory element operably linked to an enzyme-codingsequence encoding SaCas9, optionally comprising at least one or morenuclear localization sequences,

wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,

wherein components I and II are located on the same or different vectorsof the system,

wherein when transcribed, the tracr mate sequence hybridizes to thetracr sequence and the guide sequence directs sequence-specific bindingof a CRISPR complex to the target sequence, and wherein the CRISPRcomplex comprises the SaCas9 complexed with (1) the guide sequence thatis hybridized to the target sequence, and (2) the tracr mate sequencethat is hybridized to the tracr sequence,or(B) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprisingI. a first regulatory element operably linked to(a) a guide sequence capable of hybridizing to the target sequence, and(b) at least one or more tracr mate sequences,II. a second regulatory element operably linked to an enzyme-codingsequence encoding SaCas9, andIII. a third regulatory element operably linked to a tracr sequence,wherein components I, II and III are located on the same or differentvectors of the system, wherein when transcribed, the tracr mate sequencehybridizes to the tracr sequence and the guide sequence directssequence-specific binding of a CRISPR complex to the target sequence,and wherein the CRISPR complex comprises the SaCas9 complexed with (1)the guide sequence that is hybridized to the target sequence, and (2)the tracr mate sequence that is hybridized to the tracr sequence, andoptionally administering modified cells to the organism or non-humanorganism. In some methods of the invention any or all of thepolynucleotide sequence encoding the CRISPR enzyme, the first and thesecond guide sequence, the first and the second tracr mate sequence orthe first and the second tracr sequence, is/are RNA. In furtherembodiments of the invention the polynucleotides encoding the sequenceencoding the CRISPR enzyme, the first and the second guide sequence, thefirst and the second tracr mate sequence or the first and the secondtracr sequence, is/are RNA and are delivered via liposomes,nanoparticles, exosomes, microvesicles, or a gene-gun; but, it isadvantageous that the delivery is via a viral vector or a particle. Incertain embodiments of the invention, the first and second tracr matesequence share 100% identity and/or the first and second tracr sequenceshare 100% identity. In some embodiments, the polynucleotides may becomprised within a vector system comprising one or more vectors. Inpreferred embodiments of the invention the CRISPR enzyme is a Cas9enzyme, e.g. SpCas9 or SaCas9. In an aspect of the invention the CRISPRenzyme comprises one or more mutations in a catalytic domain, whereinthe one or more mutations, with reference to SpCas9 are selected fromthe group consisting of D10A, E762A, H840A, N854A, N863A and D986A,e.g., a D10A mutation. In preferred embodiments, the first CRISPR enzymehas one or more mutations such that the enzyme is a complementary strandnicking enzyme, and the second CRISPR enzyme has one or more mutationssuch that the enzyme is a non-complementary strand nicking enzyme.Alternatively the first enzyme may be a non-complementary strand nickingenzyme, and the second enzyme may be a complementary strand nickingenzyme. In preferred methods of the invention the first guide sequencedirecting cleavage of one strand of the DNA duplex near the first targetsequence and the second guide sequence directing cleavage of the otherstrand near the second target sequence results in a 5′ overhang. Inembodiments of the invention the 5′ overhang is at most 200 base pairs,preferably at most 100 base pairs, or more preferably at most 50 basepairs. In embodiments of the invention the 5′ overhang is at least 26base pairs, preferably at least 30 base pairs or more preferably 34-50base pairs.

With respect to mutations of the CRISPR enzyme, when the enzyme is notSpCas9, mutations may be made at any or all residues corresponding topositions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may beascertained for instance by standard sequence comparison tools). Inparticular, any or all of the following mutations are preferred inSpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well asconservative substitution for any of the replacement amino acids is alsoenvisaged. In an aspect the invention provides as to any or each or allembodiments herein-discussed wherein the CRISPR enzyme comprises atleast one or more, or at least two or more mutations, wherein the atleast one or more mutation or the at least two or more mutations is asto D10, E762, H840, N854, N863, or D986 according to SpCas9 protein,e.g., D10A, E762A, H840A, N854A, N863A and/or D986A as to SpCas9, orN580 according to SaCas9, e.g., N580A as to SaCas9, or any correspondingmutation(s) in a Cas9 of an ortholog to Sp or Sa, or the CRISPR enzymecomprises at least one mutation wherein at least H840 or N863A as to SpCas9 or N580A as to Sa Cas9 is mutated; e.g., wherein the CRISPR enzymecomprises H840A, or D10A and H840A, or D10A and N863A, according toSpCas9 protein, or any corresponding mutation(s) in a Cas9 of anortholog to Sp protein or Sa protein.

The invention in some embodiments comprehends a method of modifying anorganism or a non-human organism by manipulation of a first and a secondtarget sequence on opposite strands of a DNA duplex in a genomic locusof interest in a cell or cell population e.g., wherein the genomic locusof interest is associated with a mutation associated with an aberrantprotein expression or with a disease condition or state, comprisingdelivering, e.g., by contacting the cells or cell population with adelivery vector or particle(s) comprising a non-naturally occurring orengineered composition comprising:

-   -   I. a first regulatory element operably linked to        -   (a) a first guide sequence capable of hybridizing to the            first target sequence, and        -   (b) at least one or more tracr mate sequences,    -   II. a second regulatory element operably linked to        -   (a) a second guide sequence capable of hybridizing to the            second target sequence, and        -   (b) at least one or more tracr mate sequences,    -   III. a third regulatory element operably linked to an        enzyme-coding sequence encoding a CRISPR enzyme, and    -   IV. a fourth regulatory element operably linked to a tracr        sequence,    -   V. expression product(s) of one or more of I. to IV., e.g., the        the first and the second tracr mate sequence, the CRISPR enzyme;        wherein components I, II, III and IV are located on the same or        different vectors of the system, when transcribed, the tracr        mate sequence hybridizes to the tracr sequence and the first and        the second guide sequence direct sequence-specific binding of a        first and a second CRISPR complex to the first and second target        sequences respectively, wherein the first CRISPR complex        comprises the CRISPR enzyme complexed with (1) the first guide        sequence that is hybridized to the first target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence, wherein the second CRISPR complex comprises the CRISPR        enzyme complexed with (1) the second guide sequence that is        hybridized to the second target sequence, and (2) the tracr mate        sequence that is hybridized to the tracr sequence, wherein the        polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,        and wherein the first guide sequence directs cleavage of one        strand of the DNA duplex near the first target sequence and the        second guide sequence directs cleavage of the other strand near        the second target sequence inducing a double strand break,        thereby modifying the organism or the non-human organism; and        the method may optionally include also delivering a HDR        template, e.g., via the delivery vector or particle contacting        the cell or cell population containing or contacting the cell or        cell population with another particle containing, the HDR        template wherein the HDR template provides expression of a        normal or less aberrant form of the protein; wherein “normal” is        as to wild type, and “aberrant” can be a protein expression that        gives rise to a condition or disease state; and optionally the        method may include isolating or obtaining a cell or cell        population from the organism or non-human organism, optionally        expanding the cells, performing contacting of the delivery        vector or particle(s) with the cell or cell population to obtain        a modified cell population, optionally expanding the population        of modified cells, and optionally administering modified HSCs to        the organism or non-human organism.

The invention also provides a vector system as described herein. Thesystem may comprise one, two, three or four different vectors.Components I, II, III and IV may thus be located on one, two, three orfour different vectors, and all combinations for possible locations ofthe components are herein envisaged, for example: components I, II, IIIand IV can be located on the same vector; components I, II, III and IVcan each be located on different vectors; components I, II, II I and IVmay be located on a total of two or three different vectors, with allcombinations of locations envisaged, etc. In some methods of theinvention any or all of the polynucleotide sequence encoding the CRISPRenzyme, the first and the second guide sequence, the first and thesecond tracr mate sequence or the first and the second tracr sequence,is/are RNA. In further embodiments of the invention the first and secondtracr mate sequence share 100% identity and/or the first and secondtracr sequence share 100% identity. In preferred embodiments of theinvention the CRISPR enzyme is a Cas9 enzyme, e.g. SpCas9. In an aspectof the invention the CRISPR enzyme comprises one or more mutations in acatalytic domain, wherein the one or more mutations with reference toSpCas9 are selected from the group consisting of D10A, E762A, H840A,N854A, N863A and D986A; e.g., D10A mutation. In preferred embodiments,the first CRISPR enzyme has one or more mutations such that the enzymeis a complementary strand nicking enzyme, and the second CRISPR enzymehas one or more mutations such that the enzyme is a non-complementarystrand nicking enzyme. Alternatively the first enzyme may be anon-complementary strand nicking enzyme, and the second enzyme may be acomplementary strand nicking enzyme. In a further embodiment of theinvention, one or more of the viral vectors are delivered via liposomes,nanoparticles, exosomes, microvesicles, or a gene-gun; but, viraldelivery or particle delivery is advantageous

In preferred methods of the invention the first guide sequence directingcleavage of one strand of the DNA duplex near the first target sequenceand the second guide sequence directing cleavage of other strand nearthe second target sequence results in a 5′ overhang. In embodiments ofthe invention the 5′ overhang is at most 200 base pairs, preferably atmost 100 base pairs, or more preferably at most 50 base pairs. Inembodiments of the invention the 5′ overhang is at least 26 base pairs,preferably at least 30 base pairs or more preferably 34-50 base pairs.

The invention in some embodiments comprehends a method of modifying agenomic locus of interest in cell or cell population e.g., wherein thegenomic locus of interest is associated with a mutation associated withan aberrant protein expression or with a disease condition or state, byintroducing into the cell or cell population, e.g., by contacting thecells or cell population with delivery vectors or particle(s)comprising, a Cas protein having one or more mutations and two guideRNAs that target a first strand and a second strand of the DNA moleculerespectively in the cell or cell population, whereby the guide RNAstarget the DNA molecule and the Cas protein nicks each of the firststrand and the second strand of the DNA molecule, whereby a target inthe cell or cell population is altered; and, wherein the Cas protein andthe two guide RNAs do not naturally occur together and the method mayoptionally include also delivering a HDR template, e.g., via thedelivery vector or particle contacting the cell or cell populationcontaining or contacting the cell or population with another deliveryvector or particle containing the HDR template wherein the HDR templateprovides expression of a normal or less aberrant form of the protein;wherein “normal” is as to wild type, and “aberrant” can be a proteinexpression that gives rise to a condition or disease state; andoptionally the method may include isolating or obtaining cells from theorganism or non-human organism, optionally expanding the cellpopulation, performing contacting of the delivery vector or particle(s)with the cells to obtain a modified cell population, optionallyexpanding the population of modified cells and optionally administeringmodified cells to the organism or non-human organism. In preferredmethods of the invention the Cas protein nicking each of the firststrand and the second strand of the DNA molecule results in a 5′overhang. In embodiments of the invention the 5′ overhang is at most 200base pairs, preferably at most 100 base pairs, or more preferably atmost 50 base pairs. In embodiments of the invention the 5′ overhang isat least 26 base pairs, preferably at least 30 base pairs or morepreferably 34-50 base pairs. Embodiments of the invention alsocomprehend the guide RNAs comprising a guide sequence fused to a tracrmate sequence and a tracr sequence. In an aspect of the invention theCas protein is codon optimized for expression in a eukaryotic cell,preferably a mammalian cell or a human cell. In further embodiments ofthe invention the Cas protein is a type II CRISPR-Cas protein, e.g. aCas 9 protein. In a highly preferred embodiment the Cas protein is aCas9 protein, e.g. SpCas9 or SaCas9. In aspects of the invention the Casprotein has one or more mutations in respect of SpCas9 selected from thegroup consisting of D10A, E762A, H840A, N854A, N863A and D986A; e.g., aD10A mutation. Aspects of the invention relate to the expression of agene product being decreased or a template polynucleotide being furtherintroduced into the DNA molecule encoding the gene product or anintervening sequence being excised precisely by allowing the two 5′overhangs to reanneal and ligate or the activity or function of the geneproduct being altered or the expression of the gene product beingincreased. In an embodiment of the invention, the gene product is aprotein.

The invention in some embodiments comprehends a method of modifying agenomic locus of interest in a cell or cell population e.g., wherein thegenomic locus of interest is associated with a mutation associated withan aberrant protein expression or with a disease condition or state, byintroducing into the cell or cell population, e.g., by contacting thecell or cell population with a delivery vector or particle(s)comprising,

-   -   a) a first regulatory element operably linked to each of two        CRISPR-Cas system guide RNAs that target a first strand and a        second strand respectively of a double stranded DNA molecule of        the cell or cells within the cell population, and    -   b) a second regulatory element operably linked to a Cas protein,        or    -   c) expression product(s) of a) or b),        wherein components (a) and (b) are located on same or different        vectors of the system, whereby the guide RNAs target the DNA        molecule of the cells or cells within the cell population and        the Cas protein nicks each of the first strand and the second        strand of the DNA molecule of the cells or cells within the        cell; and, wherein the Cas protein and the two guide RNAs do not        naturally occur together; and the method may optionally include        also delivering a HDR template, e.g., via the delivery vector or        particle contacting the cell or cell population containing or        contacting the cell or cell population with another particle        containing, the HDR template wherein the HDR template provides        expression of a normal or less aberrant form of the protein;        wherein “normal” is as to wild type, and “aberrant” can be a        protein expression that gives rise to a condition or disease        state; and optionally the method may include isolating or        obtaining cells from the organism or non-human organism,        optionally expanding said cell population, performing contacting        of the delivery vector or particle(s) with the cells to obtain a        modified cell population, optionally expanding the population of        modified cells, and optionally administering modified cells to        the organism or non-human organism. In aspects of the invention        the guide RNAs may comprise a guide sequence fused to a tracr        mate sequence and a tracr sequence. In an embodiment of the        invention the Cas protein is a type II CRISPR-Cas protein. In an        aspect of the invention the Cas protein is codon optimized for        expression in a eukaryotic cell, preferably a mammalian cell or        a human cell. In further embodiments of the invention the Cas        protein is a type II CRISPR-Cas protein, e.g. a Cas 9 protein.        In a highly preferred embodiment the Cas protein is a Cas9        protein, e.g. SpCas9 or SaCas9. In aspects of the invention the        Cas protein has one or more mutations with reference to SpCas9        selected from the group consisting of D10A, E762A, H840A, N854A,        N863A and D986A; e.g., the D10A mutation. Aspects of the        invention relate to the expression of a gene product being        decreased or a template polynucleotide being further introduced        into the DNA molecule encoding the gene product or an        intervening sequence being excised precisely by allowing the two        5′ overhangs to reanneal and ligate or the activity or function        of the gene product being altered or the expression of the gene        product being increased. In an embodiment of the invention, the        gene product is a protein. In preferred embodiments of the        invention the vectors of the system are viral vectors. In a        further embodiment, the vectors of the system are delivered via        liposomes, nanoparticles, exosomes, microvesicles, or a        gene-gun; and particles are preferred. In one aspect, the        invention provides a method of modifying a target polynucleotide        in a cell or cell population. In some embodiments, the method        comprises allowing a CRISPR complex to bind to the target        polynucleotide to effect cleavage of said target polynucleotide        thereby modifying the target polynucleotide, wherein the CRISPR        complex comprises a CRISPR enzyme complexed with a guide        sequence hybridized to a target sequence within said target        polynucleotide, wherein said guide sequence is linked to a tracr        mate sequence which in turn hybridizes to a tracr sequence. In        some embodiments, said cleavage comprises cleaving one or two        strands at the location of the target sequence by said CRISPR        enzyme. In some embodiments, said cleavage results in decreased        transcription of a target gene. In some embodiments, the method        further comprises repairing said cleaved target polynucleotide        by homologous recombination with an exogenous template        polynucleotide, wherein said repair results in a mutation        comprising an insertion, deletion, or substitution of one or        more nucleotides of said target polynucleotide. In some        embodiments, said mutation results in one or more amino acid        changes in a protein expressed from a gene comprising the target        sequence. In some embodiments, the method further comprises        delivering one or more vectors or expression product(s) thereof,        e.g., via a delivery vector or particle(s), to said cell or cell        population, wherein the one or more vectors drive expression of        one or more of: the CRISPR enzyme, the guide sequence linked to        the tracr mate sequence, and the tracr sequence. In some        embodiments, said vectors are delivered to a cell or a cell        population in a subject. In some embodiments, said modifying        takes place in said cell or cell population in a cell culture.        In some embodiments, the method further comprises isolating said        cell or cell population from a subject prior to said modifying.        In some embodiments, the method further comprises returning said        cell or cell population and/or cells derived therefrom to said        subject.

In one aspect, the invention provides a method of generating a cell orcell population comprising a mutated disease gene. In some embodiments,a disease gene is any gene associated with an increase in the risk ofhaving or developing a disease. In some embodiments, the methodcomprises (a) introducing one or more vectors or expression product(s)thereof, e.g., via a delivery vector or particle(s), into a cell or cellpopulation, wherein the one or more vectors drive expression of one ormore of: a CRISPR enzyme, a guide sequence linked to a tracr matesequence, and a tracr sequence; and (b) allowing a CRISPR complex tobind to a target polynucleotide to effect cleavage of the targetpolynucleotide within said disease gene, wherein the CRISPR complexcomprises the CRISPR enzyme complexed with (1) the guide sequence thatis hybridized to the target sequence within the target polynucleotide,and (2) the tracr mate sequence that is hybridized to the tracrsequence, thereby generating a cell or cell population comprising amutated disease gene. In some embodiments, said cleavage comprisescleaving one or two strands at the location of the target sequence bysaid CRISPR enzyme. In some embodiments, said cleavage results indecreased transcription of a target gene. In some embodiments, themethod further comprises repairing said cleaved target polynucleotide byhomologous recombination with an exogenous template polynucleotide,wherein said repair results in a mutation comprising an insertion,deletion, or substitution of one or more nucleotides of said targetpolynucleotide. In some embodiments, said mutation results in one ormore amino acid changes in a protein expression from a gene comprisingthe target sequence. In some embodiments the modified cell or cellpopulation is administered to an animal to thereby generate an animalmodel.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a cell or cell population. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the targetpolynucleotide to effect cleavage of said target polynucleotide therebymodifying the target polynucleotide, wherein the CRISPR complexcomprises a CRISPR enzyme complexed with a guide sequence hybridized toa target sequence within said target polynucleotide, wherein said guidesequence is linked to a tracr mate sequence which in turn hybridizes toa tracr sequence. In other embodiments, this invention provides a methodof modifying expression of a polynucleotide in a eukaryotic cell thatarises from a cell or cell population expressing an aberrant protein.The method comprises increasing or decreasing expression of a targetpolynucleotide by using a CRISPR complex that binds to thepolynucleotide in the cell or cell population; advantageously the CRISPRcomplex is delivered via a viral delivery vector or particle(s).

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell or cell population. Forexample, upon the binding of a CRISPR complex to a target sequence in acell, the target polynucleotide is inactivated such that the sequence isnot transcribed, the coded protein is not produced, or the sequence doesnot function as the wild-type sequence does.

In some embodiments, the functional domain is a transcriptionalactivation domain, preferably VP64. In some embodiments, the functionaldomain is a transcription repression domain, preferably KRAB. In someembodiments, the transcription repression domain is SID, or concatemersof SID (eg SID4X). In some embodiments, the functional domain is anepigenetic modifying domain, such that an epigenetic modifying enzyme isprovided. In some embodiments, the functional domain is an activationdomain, which may be the P65 activation domain.

The invention further comprehends a composition of the invention or aCRISPR complex or enzyme thereof or RNA thereof (including oralternatively mRNA encoding the CRISPR enzyme) for use in medicine or intherapy. In some embodiments the invention comprehends a compositionaccording to the invention or components thereof for use in a methodaccording to the invention. In some embodiments the invention providesfor the use of a composition of the invention or a CRISPR complex orenzyme thereof or RNA thereof (including or alternatively mRNA encodingthe CRISPR enzyme) in ex vivo gene or genome editing, especially in acell or cell population which optionally may then be introduced into anorganism or non-human organism from which the cells or cell populationwere obtained or another organism or non-human organism of the samespecies. In certain embodiments the invention comprehends use of acomposition of the invention or a CRISPR complex or enzyme thereof orRNA thereof (including or alternatively mRNA encoding the CRISPR enzyme)in the manufacture of a medicament for ex vivo gene or genome editing orfor use in a method according of the invention. In certain embodimentsthe invention provides a method of treating or inhibiting a conditioncaused by a defect in a target sequence in a genomic locus of interestin a subject (e.g., mammal or human) or a non-human subject (e.g.,mammal) in need thereof comprising modifying a cell or a cell populationof the subject or a non-human subject by manipulation of the targetsequence in the cell or cell population and administering the modifiedcells to the subject or non-human subject, advantageously the modifyingof the cells is through contacting the cells with a delivery vector(e.g., viral) or particle containing the CRISPR complex or thecomponents thereof, advantageously in certain embodiments the deliveryvector (viral) or particle also provides a HDR template or anotherparticle or a vector provides the HDR template, and wherein thecondition is susceptible to treatment or inhibition by manipulation ofthe target sequence.

Certain RNA of the CRISPR Cas complex is also known and referred to assgRNA (single guide RNA). In advantageous embodiments RNA of the CRISPRCas complex is sgRNA. The CRISPR-Cas9 system has been engineered totarget genetic locus or loci in a cell or cell population. Cas9 protein,advantageously codon-optimized for a eukaryotic cell and especially amammalian cell, e.g., a human cell, (e.g., ocular cell, vascular cell,cochclear cell, etc.) and sgRNA targeting a locus or loci in the cell,e.g., the gene RHO, ATOH1, VEGFA were prepared, and are exemplifiedherein. These were advantageously delivered via a viral delivery (AAV).When delivered via particles, the particles are formed by the Cas9protein and the sgRNA being admixed. The sgRNA and Cas9 protein mixtureis admixed with a mixture comprising or consisting essentially of orconsisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol, whereby particles containing the sgRNA and Cas9protein are formed. The invention comprehends so making particles andparticles from such a method as well as uses thereof. More generally,particles were formed using an efficient process. First, Cas9 proteinand sgRNA targeting a gene or a control gene LacZ are mixed together ata suitable, e.g., 3:1 to 1:3 or 2:1 to 1:2 or 1:1 molar ratio, at asuitable temperature, e.g., 15-30 C, e.g., 20-25 C, e.g., roomtemperature, for a suitable time, e.g., 15-45, such as 30 minutes,advantageously in sterile, nuclease free buffer, e.g., 1×PBS.Separately, particle components such as or comprising: a surfactant,e.g., cationic lipid, e.g., 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP); phospholipid, e.g., dimyristoylphosphatidylcholine (DMPC);biodegradable polymer, such as an ethylene-glycol polymer or PEG, and alipoprotein, such as a low-density lipoprotein, e.g., cholesterol weredissolved in an alcohol, advantageously a C₁₋₆ alkyl alcohol, such asmethanol, ethanol, isopropanol, e.g., 100% ethanol. The two solutionsare mixed together to form particles containing the Cas9-sgRNAcomplexes. In certain embodiments the particle can contain an HDRtemplate. That can be a particle co-administered with sgRNA+Cas9protein-containing particle, or i.e., in addition to contacting a cellor cell population with an sgRNA+Cas9 protein-containing particle, thecell or cell population is contacted with a particle containing an HDRtemplate; or the HSC is contacted with a particle containing all of thesgRNA, Cas9 and the HDR template. The HDR template can be administeredby a separate vector, whereby in a first instance the particlepenetrates an HSC cell and the separate vector also penetrates the cell,wherein the HSC genome is modified by the sgRNA+Cas9 and the HDRtemplate is also present, whereby a genomic loci is modified by the HDR;for instance, this may result in correcting a mutation. The particle inthe herein discussion is advantageously obtained or obtainable fromadmixing an sgRNA(s) and Cas9 protein mixture (optionally containing HDRtemplate(s) or such mixture only containing HDR template(s) whenseparate particles as to template(s) is desired) with a mixturecomprising or consisting essentially of or consisting of surfactant,phospholipid, biodegradable polymer, lipoprotein and alcohol (whereinone or more sgRNA targets agenetic locus or loci associated with amutation associated with an aberrant protein xpression or with a diseasecondition or state).

In one aspect, the invention provides for methods of modeling a diseaseassociated with a genomic locus in a eukaryotic organism or a non-humanorganism comprising manipulation of a target sequence within a coding,non-coding or regulatory element of said genomic locus comprisingdelivering a non-naturally occurring or engineered compositioncomprising:

-   -   (A)—I. a CRISPR-Cas system RNA polynucleotide sequence, wherein        the polynucleotide sequence comprises:        -   (a) a guide sequence capable of hybridizing to the target            sequence,        -   (b) a tracr mate sequence, and        -   (c) a tracr sequence, and    -   II. a polynucleotide sequence encoding Cas9, optionally        comprising at least one or more nuclear localization sequences,        wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises Cas9 complexed with (1) the        guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence and the polynucleotide sequence encoding Cas9 is DNA or        RNA, or    -   (B) I. polynucleotides comprising:        -   (a) a guide sequence capable of hybridizing to the target            sequence, and        -   (b) at least one or more tracr mate sequences,    -   II. a polynucleotide sequence encoding Cas9, and    -   III. a polynucleotide sequence comprising a tracr sequence,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the Cas9 complexed with (1)        the guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence, and the polynucleotide sequence encoding Cas9 is DNA        or RNA.

In certain preferred embodiments, the Cas9 is SaCas9.

In one aspect, the invention provides for methods of modeling a diseaseassociated with a genomic locus in a eukaryotic organism or a non-humanorganism comprising manipulation of a target sequence within a coding,non-coding or regulatory element of said genomic locus comprisingdelivering a non-naturally occurring or engineered compositioncomprising a viral vector system comprising one or more viral vectorsoperably encoding a composition for expression thereof, wherein thecomposition comprises:

-   -   (A) a non-naturally occurring or engineered composition        comprising a vector system comprising one or more vectors        comprising    -   I. a first regulatory element operably linked to a CRISPR-Cas        system RNA polynucleotide sequence, wherein the polynucleotide        sequence comprises        -   (a) a guide sequence capable of hybridizing to the target            sequence,        -   (b) a tracr mate sequence, and        -   (c) a tracr sequence, and    -   II. a second regulatory element operably linked to an        enzyme-coding sequence encoding Cas9, (preferably SaCas9)        optionally comprising at least one or more nuclear localization        sequences,        wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein components I and II are located on the same or different        vectors of the system,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the Cas9 complexed with (1)        the guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence,        or    -   (B) a non-naturally occurring or engineered composition        comprising a vector system comprising one or more vectors        comprising    -   I. a first regulatory element operably linked to        -   (a) a guide sequence capable of hybridizing to the target            sequence, and        -   (b) at least one or more tracr mate sequences,    -   II. a second regulatory element operably linked to an        enzyme-coding sequence encoding Cas9, and    -   III. a third regulatory element operably linked to a tracr        sequence,        wherein components I, II and III are located on the same or        different vectors of the system,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the Cas9 complexed with (1)        the guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence.

In one aspect the invention provides methods of treating or inhibiting acondition or a disease caused by one or more mutations in a genomiclocus in a eukaryotic organism or a non-human organism comprisingmanipulation of a target sequence within a coding, non-coding orregulatory element of said genomic locus in a target sequence in asubject or a non-human subject in need thereof comprising modifying thesubject or a non-human subject by manipulation of the target sequenceand wherein the condition or disease is susceptible to treatment orinhibition by manipulation of the target sequence comprising providingtreatment comprising:

delivering a non-naturally occurring or engineered compositioncomprising an AAV or lentivirus vector system, comprising one or moreAAV or lentivirus vectors operably encoding a composition for expressionthereof, wherein the target sequence is manipulated by the compositionwhen expressed, wherein the composition comprises:

-   -   (A) a non-naturally occurring or engineered composition        comprising a vector system comprising one or more vectors        comprising    -   I. a first regulatory element operably linked to a CRISPR-Cas        system RNA polynucleotide sequence, wherein the polynucleotide        sequence comprises        -   (a) a guide sequence capable of hybridizing to the target            sequence in a eukaryotic cell,        -   (b) a tracr mate sequence, and        -   (c) a tracr sequence, and    -   II. a second regulatory element operably linked to an        enzyme-coding sequence encoding Cas9, preferably SaCas9,        comprising at least one or more nuclear localization sequences,        wherein (A), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein components I and II are located on the same or different        vectors of the system,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the Cas9 complexed with (1)        the guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence, or    -   (B) a non-naturally occurring or engineered composition        comprising a vector system comprising one or more vectors        comprising    -   I. a first regulatory element operably linked to        -   (a) a guide sequence capable of hybridizing to an target            sequence in a eukaryotic cell, and        -   (b) at least one or more tracr mate sequences,    -   II. a second regulatory element operably linked to an        enzyme-coding sequence encoding Cas9, preferably SaCas9, and    -   III. a third regulatory element operably linked to a tracr        sequence, wherein components I, II and III are located on the        same or different vectors of the system, wherein when        transcribed, the tracr mate sequence hybridizes to the tracr        sequence and the guide sequence directs sequence-specific        binding of a CRISPR complex to the target sequence, and wherein        the CRISPR complex comprises Cas9 complexed with (1) the guide        sequence that is hybridized to the target sequence, and (2) the        tracr mate sequence that is hybridized to the tracr sequence.

In certain embodiments, the invention provides method of preparing theAAV or lentivirus vector for use in accordance with any of the methodsof the invention, comprising transfecting plasmid(s) containing orconsisting essentially of nucleic acid molecule(s) coding for the AAV orlentivirus into AAV-infected or lentivirus-infected cells, and supplyingAAV AAV or lentivirus rep and/or cap and/or helper nucleic acidmolecules obligatory for replication and packaging of the AAV orlentivirus.

In one aspect, the invention provides a composition for use in any ofthe methods of invention (e.g., method of modeling a disease associatedwith a genetic locus in a eukaryotic organism or a non-human organism)comprising manipulation of a target sequence within a coding, non-codingor regulatory element of said genetic locus. In certain embodiments, theinvention provides for uses of the composition in ex vivo or in vivogene or genome editing, including therapeutic uses.

In one aspect, the invention provides a composition for use in themanufacture of a medicament for in vitro, ex vivo or in vivo gene orgenome editing or for use in a method of modifying an organism or anon-human organism by manipulation of a target sequence in a genomiclocus associated with a disease or in a method of treating or inhibitinga condition or disease caused by one or more mutations in a genomiclocus in a eukaryotic organism or a non-human organism.

In one aspect, the invention provides a composition comprising:

-   -   (A)—I. a CRISPR-Cas system RNA polynucleotide sequence, wherein        the polynucleotide sequence comprises:        -   (a) a guide sequence capable of hybridizing to a target            sequence in a eukaryotic cell,        -   (b) a tracr mate sequence, and        -   (c) a tracr sequence, and    -   II. a polynucleotide sequence encoding Cas9, preferably Sa Cas9,        optionally comprising at least one or more nuclear localization        sequences,        wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the Cas9 complexed with (1)        the guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence and the polynucleotide sequence encoding Cas9 is DNA or        RNA,        or    -   (B) I. polynucleotides comprising:        -   (a) a guide sequence capable of hybridizing to an target            sequence in a eukaryotic cell, and        -   (b) at least one or more tracr mate sequences,    -   II. a polynucleotide sequence encoding Cas9, preferably SaCas9,        and    -   III. a polynucleotide sequence comprising a tracr sequence,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises SaCas9 complexed with (1)        the guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence, and the polynucleotide sequence encoding Cas9 is DNA        or RNA;        for use in medicine or therapy; or for use in a method of        modifying an organism or a non-human organism by manipulation of        a target sequence in a genomic locus associated with a disease        or disorder; or for use in a method of treating or inhibiting a        condition caused by one or more mutations in a genetic locus        associated with a disease in a eukaryotic organism or a        non-human organism; or for use in in vitro, ex vivo or in vivo        gene or genome editing.

In one aspect, the invention provides a therapeutic genome editingmethod for treating or inhibiting a condition or a disease caused by oneor more mutations in a genomic locus in a eukaryotic organism or anon-human organism comprising manipulation of a target sequence within acoding, non-coding or regulatory element of said genomic locus in atarget sequence in a subject or a non-human subject in need thereofcomprising modifying the subject or a non-human subject by manipulationof the target sequence and wherein the condition or disease issusceptible to treatment or inhibition by manipulation of the targetsequence comprising providing treatment comprising:

delivering a non-naturally occurring or engineered compositioncomprising an AAV or lentivirus vector system, comprising one or moreAAV or lentivirus vectors operably encoding a composition for expressionthereof, wherein the target sequence is manipulated by the compositionwhen expressed, wherein the composition comprises:

(A) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising

I. a first regulatory element operably linked to a CRISPR-Cas system RNApolynucleotide sequence, wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to the target sequence in aeukaryotic cell,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a second regulatory element operably linked to an enzyme-codingsequence encoding Cas9, preferably SaCas9, comprising at least one ormore nuclear localization sequences,

wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,

wherein components I and II are located on the same or different vectorsof the system,

wherein when transcribed, the tracr mate sequence hybridizes to thetracr sequence and the guide sequence directs sequence-specific bindingof a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the Cas9 complexed with (1) theguide sequence that is hybridized to the target sequence, and (2) thetracr mate sequence that is hybridized to the tracr sequence,

or

(B) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising

I. a first regulatory element operably linked to

(a) a guide sequence capable of hybridizing to an target sequence in aeukaryotic cell, and

(b) at least one or more tracr mate sequences,

II. a second regulatory element operably linked to an enzyme-codingsequence encoding SaCas9, and

III. a third regulatory element operably linked to a tracr sequence,

wherein components I, II and III are located on the same or differentvectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to thetracr sequence and the guide sequence directs sequence-specific bindingof a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises Cas9 complexed with (1) the guidesequence that is hybridized to the target sequence, and (2) the tracrmate sequence that is hybridized to the tracr.

In one aspect, the invention provides a method of individualized orpersonalized treatment of a genetic disease in a subject in need of suchtreatment comprising:

(a) introducing multiple mutations ex vivo in a tissue, organ or a cellline comprising Cas9-expressing eukaryotic cell(s) (preferably Sa Cas9),or in vivo in a transgenic non-human mammal having cells that expressCas9, comprising delivering to cell(s) of the tissue, organ, cell ormammal the vector as herein-discussed, wherein the specific mutations orprecise sequence substitutions are or have been correlated to thegenetic disease;

(b) testing treatment(s) for the genetic disease on the cells to whichthe vector has been delivered that have the specific mutations orprecise sequence substitutions correlated to the genetic disease; and

(c) treating the subject based on results from the testing oftreatment(s) of step (b).

In certain embodiments of any of the aforementioned aspects andembodiments of the invention, the viral vector may be an AAV, e.g.,AAV1, AAV2, AAV5, AAV7, AAV8, AAV DJ or any combination thereof.

In herein discussions concerning the target being associated with amutation or with a disease condition, such mutation or disease conditioncan be, for instance a neuronal disease; ocular disease (e.g., retinadisease, e.g., retinitis pigmentosa; achromtaopsia; age-related maculardegeneration; visual impairment), auditory disease (e.g., cochlear-cellassociated disease, hearing impairment, deafness) etc.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like,and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention. It may be advantageous in the practiceof the invention to be in compliance with Art. 53(c) EPC and Rule 28(b)and (c) EPC. There are no promises in this document.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A-H shows CRISPR-Cas9 system delivery and targeting of Mecp2 locusin the mouse brain. (a) AAV-SpCas9 and AAV-SpGuide(Mecp2) expressionvectors. The sgRNA vector contains encoding sequence of the GFP-KASHfusion protein for identification of transduced neurons. (b) Expressionof HA-Cas9 and GFP-KASH in the dorsal dentate gyrus (DG) of mousehippocampus. Scale bar, 100 μm. (c) Quantification of cells efficientlytargeted by the dual-vector Cas9-CRISPR system. (d) Graphicalrepresentation of the mouse Mecp2 locus showing Cas9 target location:sgRNA indicated in blue. PAM sequence marked in purple. Representativemutation patterns detected by sequencing of Mecp2 locus were shownbelow: green—wild-type sequence; red dashes—deleted bases; red bases:insertion or mutations; red arrowhead indicates CRISPR-Cas9 cutting site(SEQ ID NOS 127-141, respectively, in order of appearance). (e)SURVEYOR™ assay gel showing modification of the Mecp2 locus, 2 weeksafter AAV delivery in the DG region. (f) Western blot analysis of MeCP2protein expression in the targeted brain region and quantification ofMeCP2 protein levels in dorsal DG (t-test, **p<0.001, n=4 from 3animals, error bars: s.e.m.). (g) Images of the dorsal DG region, 2weeks after CRISPR-Cas9 targeting of Mecp2 locus. Scale bar, 150 μm. (h)Quantification of MeCP2 positive cells population within all detectedcells (DAPI staining) in the targeted brain region in compare to controlcollateral site (Q-test, ****p<0.0001, n=290 and 249 cells from 2animals, respectively; error bars: s.e.m). (ITR—inverted terminalrepeat; HA—hemagglutinin tag; NLS—nuclear localization signal;spA—synthetic polyadenylation signal; U6—PolIII promoter; sgRNA—singleguide RNA; hSyn—human synapsin 1 promoter; GFP—green fluorescentprotein; KASH—Klarsicht, ANC1, Syne Homology nuclear transmembranedomain; bGH pA—bovine growth hormone polyadenylatio signal,WPRE—Woodchuck Hepatitis virus posttranscriptional regulatory element).

FIG. 2A-B shows analysis of gene expression in Cas9-mediated MCCP2knockdown neurons. (a) Strategy for cell nuclei purification ofCRISPR-Cas9 targeted cells from the mouse brain. (b) Hierarchicalclustering of differentially expressed genes (Q-test, p<0.01, n=19populations of sorted nuclei from 8 animals) detected by RNAseq.Relative log 2(TPM+1) expression levels of genes are normalized for eachrow and displayed in red-blue color scale. Each column represents apopulation of targeted 100 neuronal nuclei FACS sorted from theisolated, dentate gyrus population of cells, either from control orMecp2 sgRNA transduced animals, as indicated.

FIG. 3A-E shows cell-autonomous defects in cellular response propertiesof neurons after CRISPR-mediated MeCP2 knockdown. (a) Cartoon showing invivo experiment configuration from mouse visual cortex and visualstimulation parameter. GFP⁺ neuron is shown. Scale bar, 20 μm. (b)Cartoon showing recording configuration in layer 2/3 excitatory neuronsthat receive both contra- and ipsilateral eye specific input. Genomemodified GFP⁺ cells are in green whereas unmodified cells are in gray.Normalized spike shape shows regular spiking excitatory neurons. (c,d)Average OSI (c) and evoked FR (d) were measured from GFP⁺ cellsexpressing Mecp2 and control sgRNA, respectively (t-test, *p<0.05;numbers in graph indicate numbers of recorded cells; n=2-3 animals;error bars: s.e.m).

FIG. 4A-F shows simultaneous, multiplex gene editing in the mouse brain.(a) Schematic illustration of CRISPR-Cas9 system designed for multiplexgenome targeting. (b) Graphical representation of targeted DNMT mouseloci. Guide RNAs are indicated in blue. PAM sequences are marked inpurple (SEQ ID NOS 142-147, respectively, in order of appearance). (c)SURVEYOR™ assay gel showing modification of DNMTs loci in FACS sortedGFP-KASH positive cells, 4 weeks after AAV delivery in the DG region.(d) Deep sequencing-based analysis of DNMTs loci modification in singlecells, showing co-occurrence of modification in multiple loci. (e)Western blot analysis for Dnmt3a and Dnmt1 proteins after in vivodelivery of CRISPR-Cas 9 system targeting DNMT family genes (top).Western blot quantification of Dnmt3a and Dnmt1 protein levels in DGafter in vivo CRISPR-Cas9 targeting (bottom; t-test, **p<0.001, *p<0.05,Dnmt3a: n=7; Dnmt1: n=5 from 5 animals; error bars: s.e.m). (f)Contextual learning deficits, 8 weeks after targeting of DNMT genesusing SpCas9 in the DG region of hippocampus, tested in training andaltered context (t-test, ***p<0.0001, n=18 animals, 2 independentexperiments; error bars: s.e.m).

FIG. 5A-F shows cloning and expression of HA-tagged SpCas9 (HA-SpCas9)for AAV packaging. (a) Schematic overview of different cloningstrategies to minimize SpCas9 expression cassette size using short ratMap1b promotor (pMap1b), a truncated version of the mouse Mecp2 promoter(pMecp2) and a short polyA motif (spA). (b) Western blot analysis ofprimary cortical neuron culture expressing HA-SpCas9 using differentSpCas9 expression cassettes. (c) Mecp2 promoter drives HA-SpCas9 (red)expression in neurons (Map1b, NeuN; arrows) but not in astroglia (GFAP,arrowheads). Co-expression of HA-SpCas9 with GFP-KASH is shown (bottom).Nuclei were labeled with DAPI (blue). Scale bars, 20 μm. (d) Schematicoverview of GFP-labeling. Enhanced green fluorescent protein (GFP) fusedto the nuclear transmembrane KASH domain and integration of GFP-KASH tothe outer nuclear membrane is illustrated. (e) Co-infection efficiencycalculation, showing populations of cell expressing both HA-SpCas9 andGFP-KASH (n=973 neurons from 3 cultures; error bars: s.e.m). (f) Cellswere stained with LIFE/DEAD® kit 7 days after virus delivery.Quantification of DAPI⁺ and dead (DEAD⁺) cells (control n=518 DAPI⁺nuclei; SpCas9/GFP-KASH n=1003 DAPI⁺ nuclei from 2 cultures; error bars:s.e.m). (ITR—inverted terminal repeat; HA—hemagglutinin tag; NLS—nuclearlocalization signal; spA—synthetic polyadenylation signal; U6—PolIIIpromoter; sgRNA—single guide RNA; hSyn—human synapsin 1 promoter;GFP—green fluorescent protein; KASH—Klarsicht, ANC1, Syne Homologynuclear transmembrane domain; bGH pA—bovine growth hormonepolyadenylation signal; WPRE—Woodchuck Hepatitis virusposttranscriptional regulatory element).

FIG. 6A-B shows targeting of Mecp2 in Neuro-2a cells. (a) Mecp2targeting sequences and corresponding protospacer adjacent motifs (PAM)(SEQ ID NOS 148-151, 129 and 152, respectively, in order of appearance).(b) Evaluation of 6 Mecp2 sgRNAs co-transfected with SpCas9 intoNeuro-2a cells. Locus modification efficiencies were analyzed 48 h aftertransfection using SURVEYOR™ assay.

FIG. 7A-D shows CRISPR-SpCas9 targeting of Mecp2 in primary corticalneurons.

-   -   (a) Immunofluorescent staining of MeCP2 (red) in cultured        neurons 7 days after AAV-CRISPR transduction (green, GFP-KASH).        Nuclei were labeled with DAPI (blue). Scale bar, 20 μm. (b)        Evaluation of Mecp2 locus targeting using SpCas9 or dSpCas9,        together with Mecp2 sgRNA or control (targeting bacterial lacZ        gene) sgRNA, using SURVEYOR™ assay gel. (c) Quantification of        MeCP2 positive nuclei in targeted population of neurons        (GFP⁺). (d) Western blot of MeCP2 protein levels after        CRISPR-SpCas9 targeting of Mecp2 locus and quantification of        MeCP2 protein levels (t-test, **p<0.001, n=5 from 3 cultures,        error bars: s.e.m).

FIG. 8A-E shows morphological changes in dendritic tree of neurons afterSpCas9-mediated MeCP2 knockdown in vitro. (a) Reduced complexity ofdendritic tree in neurons after CRISPR-SpCas9 targeting of Mecp2 locus.Scale bar, 20 μm. (b) Changes in dendritic spines morphology in neuronstargeted with SpCas9 and Mecp2 sgRNA. Scale bar, 10 μm. Morphology ofcells was visualized with co-transfection with mCherry construct. Cellsfor morphology analysis were chosen based on the result of Mecp2staining. (c) Dendritic tree morphology assessed with number ofdendritic ends and (d) Sholl analysis (t-test, ***p<0.0001, n=40 from 2cultures). (e) Spine density quantification (t-test, ***p<0.0001, n=40from 2 cultures, error bars: s.e.m).

FIG. 9 shows RNAseq of neuronal nuclei from control animals andSpCas9-mediated Mecp2 knockdown. Box plot presenting the number ofdetected genes across the RNA-seq libraries (19 libraries each of 100nuclei taken from control sgRNA or Mecp2 sgRNA transduced nuclei; n=4animals/group) per quantile of expression level. All genes are dividedto 10 quantiles by their mean log 2(TPM+1) expression level, then foreach quantile the number of genes that are detected (log 2(TPM+1)>2) wascounted in each sample. The three target sequences shown are SEQ ID NO:1, SEQ ID NO: 2 and SEQ ID NO: 3, for Dnmt3a, Dnmt1 and Dnmt3b,respectively.

FIG. 10A-B shows multiplex genome targeting of DNMT family members invitro. (a) DnmI3a (SEQ ID NO: 153), Dnmt1 (SEQ ID NO: 154) and Dnmt3b(SEQ ID NO: 155) targeting sequences and corresponding protospaceradjacent motifs (PAM). (b) SURVEYOR™ nuclease assay analysis of Neuro-2acells 48 hours after transfection with SpCas9 and DNMT 3×sgRNA vectortargeting Dnmt3a, Dnmt1 and Dnmt3b loci. Efficient genome editing of allthree targeted genes is shown.

FIG. 11A-C shows next generation sequencing of targeted Dnmt3a, Dnmt1and Dnmt3b loci. Examples of sequencing results of mutated Dnmt3a (a)(SEQ ID NOS 156-163, 161 and 164-166, respectively, in order ofappearance), Dnmt1 (b) (SEQ ID NOS 167-171, 170, 172-173, 172, 170 and174-175, respectively, in order of appearance) and Dnmt3b (c) (SEQ IDNOS 155 and 176-183, respectively, in order of appearance) loci after invivo delivery of SpCas9 and DNMT 3×sgRNA into the mouse dentate gyrus.Green: wild-type sequence, red dashes: deleted bases, red bases:insertion or mutations. Red arrowheads indicate CRISPR-SpCas9 cuttingsite. The full sequences used in this figure are provide as SEQ ID NO:1, SEQ ID NO: 2, and SEQ ID NO: 3 for the Dnmt3a, the Dnmt1 and theDnmt3b loci, respectively. They are: SEQ ID NO: 1 (Dnmt3a): CCT CCG TGTCAG CGA CCC ATG CCA A, SEQ ID NO: 2 (Dnmt1): CCA GCG TCG AAC AGC TCC AGCCCG and SEQ ID NO: 3 (Dnmt3b) AGA GGG TGC CAG CGG GTA TAT GAG G.

FIG. 12 shows comparison of different programmable nuclease platforms.

FIG. 13A-C show types of therapeutic genome modifications. The specifictype of genome editing therapy depends on the nature of the mutationcausing disease. a, In gene disruption, the pathogenic function of aprotein is silenced by targeting the locus with NHEJ. Formation ofindels on the gene of interest often result in frameshift mutations thatcreate premature stop codons and a non-functional protein product, ornon-sense mediated decay of transcripts, suppressing gene function. b,HDR gene correction can be used to correct a deleterious mutation. DSBis targeted near the mutation site in the presence of an exogenouslyprovided, corrective HDR template. HDR repair of the break site with theexogenous template corrects the mutation, restoring gene function. c, Analternative to gene correction is gene addition. This mode of treatmentintroduces a therapeutic transgene into a safe-harbor locus in thegenome. DSB is targeted to the safe-harbor locus and an HDR templatecontaining homology to the break site, a promoter and a transgene isintroduced to the nucleus. HDR repair copies the promoter-transgenecassette into the safe-harbor locus, recovering gene function, albeitwithout true physiological control over gene expression.

FIG. 14 shows a schematic representation of ex vivo vs. in vivo editingtherapy. In ex vivo editing therapy cells are removed from a patient,edited and then re-engrafted (top panel). For this mode of therapy to besuccessful, target cells must be capable of survival outside the bodyand homing back to target tissues post-transplantation. In vivo therapyinvolves genome editing of cells in situ (bottom panels). For in vivosystemic therapy, delivery agents that are relatively agnostic to cellidentity or state would be used to effect editing in a wide range oftissue types. Although this mode of editing therapy may be possible inthe future, no delivery systems currently exist that are efficientenough to make this feasible. In vivo targeted therapy, where deliveryagents with tropism for specific organ systems are administered topatients are feasible with clinically relevant viral vectors.

FIG. 15 shows SaCas9 system for ocular gene therapy

FIG. 16 shows a schematic representation of gene therapy via Cas9Homologous Recombination (HR) vectors.

FIG. 17 shows an exemplary protocol for ocular gene therapy.

FIG. 18A-B show the human RHO locus (allele showing P23H mutation). FIG.18A shows the guide design for RHO locus (SEQ ID NO: 184). FIG. 18Bshows the in vitro guide screening results using the SURVEYOR assay.

FIG. 19 shows RHO HR AAV vector.

FIG. 20A-B shows guide selection for CNGA3 and CNGB3. (a) shows humanCNGA3 locus (allele showing two disease mutations) and guide selection(SEQ ID NO: 185). (b) shows human CNGB3 locus (allele showing diseasemutation) and guide selection (SEQ ID NO: 186).

FIG. 21 shows CNGA3 HR AAV vector.

FIG. 22 shows CNGB3 HR AAV vector.

FIG. 23A-B show guide selection for VEGFA. (a) shows human VEGFA lcous(common region 1) (SEQ ID NO: 187); (b) shows human VEGFA lcous (commonregion 2) (SEQ ID NO: 188).

FIG. 24 shows design of dCas9-based epigenetic modulation system (3components of the system, dSaCas9, fusion effector, and sgRNA areshown).

FIG. 25A-C shows guide selection for ATOH1. (a) shows two highlyaccessible regions which were selected; (b) shows highly accessibleregion 1-blue lines indicate guide sequence and magenta lines indicatePAM (SEQ ID NOS 189, 195 and 190, respectively, in order of appearance);(c) shows highly accessible region 2-blue lines indicate guide sequenceand magenta lines indicate PAM (SEQ ID NOS 191-192, respectively, inorder of appearance).

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

With respect to general information on CRISPR-Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, AAV, and making and usingthereof, including as to amounts and formulations, all useful in thepractice of the instant invention, reference is made to: U.S. Pat. Nos.8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356,8,889,418 and 8,895,308; US Patent Publications US 2014-0310830 (U.S.application Ser. No. 14/105,031), US 2014-0287938 A1 (U.S. applicationSer. No. 14/213,991), US 2014-0273234 A1 (U.S. application Ser. No.14/293,674), US2014-0273232 A1 (U.S. application Ser. No. 14/290,575),US 2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046A1 (U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. No. 14/222,930), US 2014-0242699 A1 (U.S. application Ser. No.14/183,512), US 2014-0242664 A1 (U.S. application Ser. No. 14/104,990),US 2014-0234972 A1 (U.S. application Ser. No. 14/183,471), US2014-0227787 A1 (U.S. application Ser. No. 14/256,912), US 2014-0189896A1 (U.S. application Ser. No. 14/105,035), US 2014-0186958 (U.S.application Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. applicationSer. No. 14/104,977), US 2014-0186843 A1 (U.S. application Ser. No.14/104,900), US 2014-0179770 A1 (U.S. application Ser. No. 14/104,837)and US 2014-0179006 A1 (U.S. application Ser. No. 14/183,486), US2014-0170753 (U.S. application Ser. No. 14/183,429); European PatentApplications EP 2 771 468 (EP13818570.7), EP 2 764 103 (EP13824232.6),and EP 2 784 162 (EP14170383.5); and PCT Patent Publications WO2014/093661 (PCT/US2013/074743), WO 2014/093694 (PCT/US2013/074790), WO2014/093595 (PCT/US2013/074611), WO 2014/093718 (PCT/US2013/074825), WO2014/093709 (PCT/US2013/074812), WO 2014/093622 (PCT/US2013/074667), WO2014/093635 (PCT/US2013/074691), WO 2014/093655 (PCT/US2013/074736), WO2014/093712 (PCT/US2013/074819), WO2014/093701 (PCT/US2013/074800), andWO2014/018423 (PCT/US2013/051418). Reference is also made to U.S.provisional patent applications 61/758,468; 61/802,174; 61/806,375;61/814,263; 61/819,803 and 61/828,130, filed on Jan. 30, 2013; Mar. 15,2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013respectively. Reference is also made to U.S. provisional patentapplication 61/836,123, filed on Jun. 17, 2013. Reference isadditionally made to U.S. provisional patent applications 61/835,931,61/835,936, 61/836,127, 61/836,101, 61/836,080 and 61/835,973, eachfiled Jun. 17, 2013. Further reference is made to U.S. provisionalpatent applications 61/862,468 and 61/862,355 filed on Aug. 5, 2013;61/871,301 filed on Aug. 28, 2013; 61/960,777 filed on Sep. 25, 2013 and61/961,980 filed on Oct. 28, 2013. Reference is yet further made to: PCTPatent applications Nos: PCT/US2014/041803, PCT/US2014/041800,PCT/US2014/041809, PCT/US2014/041804 and PCT/US2014/041806, each filedJun. 10, 2014 6/10/14; PCT/US2014/041808 filed Jun. 11, 2014; andPCT/US2014/62558 filed Oct. 28, 2014, and U.S. Provisional PatentApplications Ser. Nos. 61/915,150, 61/915,301, 61/915,267 and61/915,260, each filed Dec. 12, 2013; 61/757,972 and 61/768,959, filedon Jan. 29, 2013 and Feb. 25, 2013; 61/835,936, 61/836,127, 61/836,101,61/836,080, 61/835,973, and 61/835,931, filed Jun. 17, 2013; 62/010,888and 62/010,879, both filed Jun. 11, 2014; 62/010,329 and 62/010,441,each filed Jun. 10, 2014; 61/939,228 and 61/939,242, each filed Feb. 12,2014; 61/980,012, filed Apr. 15, 2014; 62/038,358, filed Aug. 17, 2014;62/054,490, 62/055,484, 62/055,460 and 62/055,487, each filed Sep. 25,2014; and 62/069,243, filed Oct. 27, 2014. Reference is also made toU.S. provisional patent applications Nos. 62/055,484, 62/055,460, and62/055,487, filed Sep. 25, 2014; U.S. provisional patent application61/980,012, filed Apr. 15, 2014; and U.S. provisional patent application61/939,242 filed Feb. 12, 2014. Reference is made to PCT applicationdesignating, inter alia, the United States, application No.PCT/US14/41806, filed Jun. 10, 2014. Reference is made to U.S.provisional patent application 61/930,214 filed on Jan. 22, 2014.Reference is made to U.S. provisional patent applications 61/915,251;61/915,260 and 61/915,267, each filed on Dec. 12, 2013. Reference ismade to U.S. provisional patent application U.S. Ser. No. 61/980,012filed Apr. 15, 2014. Reference is made to PCT application designating,inter alia, the United States, application No. PCT/US14/41806, filedJun. 10, 2014. Reference is made to U.S. provisional patent application61/930,214 filed on Jan. 22, 2014. Reference is made to U.S. provisionalpatent applications 61/915,251; 61/915,260 and 61/915,267, each filed onDec. 12, 2013. Each of these patents, patent publications, andapplications, and all documents cited therein or during theirprosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, together with any instructions,descriptions, product specifications, and product sheets for anyproducts mentioned therein or in any document therein and incorporatedby reference herein, are hereby incorporated herein by reference, andmay be employed in the practice of the invention. All documents (e.g.,these patents, patent publications and applications and the appln citeddocuments) are incorporated herein by reference to the same extent as ifeach individual document was specifically and individually indicated tobe incorporated by reference.

Also with respect to general information on CRISPR-Cas Systems, mentionis made of the following (also hereby incorporated herein by reference):

-   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,    Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D.,    Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science February    15; 339(6121):819-23 (2013);-   RNA-guided editing of bacterial genomes using CRISPR-Cas systems.    Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A. Nat Biotechnol    March; 31(3):233-9 (2013);-   One-Step Generation of Mice Carrying Mutations in Multiple Genes by    CRISPR/Cas-Genome Engineering. Wang H., Yang H., Shivalila C S.,    Dawlaty M M., Cheng A W., Zhang F., Jaenisch R. Cell May 9;    153(4):910-8 (2013);-   Optical control of mammalian endogenous transcription and epigenetic    states. Konermann S, Brigham M D, Trevino A E, Hsu P D, Heidenreich    M, Cong L, Platt R J, Scott D A, Church G M, Zhang F. Nature. 2013    Aug. 22; 500(7463):472-6. doi: 10.1038 Nature 12466. Epub 2013 Aug.    23;-   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing    Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J S.,    Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba, S.,    Zhang, Y., & Zhang, F. Cell August 28. pii: S0092-8674(13)01015-5.    (2013);-   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,    Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V.,    Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L    A., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013);-   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P    D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature    Protocols November; 8(11):2281-308. (2013);-   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem,    O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson,    T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F.    Science December 12. (2013). [Epub ahead of print];-   Crystal structure of cas9 in complex with guide RNA and target DNA.    Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S., Shehata, S I.,    Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell February 27.    (2014). 156(5):935-49;-   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian    cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon D    B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch R.,    Zhang F., Sharp P A. Nat Biotechnol. (2014) April 20. doi:    10.1038/nbt.2889,-   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling,    Platt et al., Cell 159(2): 440-455 (2014) DOI:    10.1016/j.cell.2014.09.014,-   Development and Applications of CRISPR-Cas9 for Genome Engineering,    Hsu et al, Cell 157, 1262-1278 (Jun. 5, 2014) (Hsu 2014),-   Genetic screens in human cells using the CRISPR/Cas9 system, Wang et    al., Science. 2014 Jan. 3; 343(6166): 80-84.    doi:10.1126/science.1246981,-   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated    gene inactivation, Doench et al., Nature Biotechnology published    online 3 Sep. 2014; doi:10.1038/nbt.3026, and-   In vivo interrogation of gene function in the mammalian brain using    CRISPR-Cas9, Swiech et al, Nature Biotechnology; published online 19    Oct. 2014;    -   doi:10.1038/nbt.3055.        each of which is incorporated herein by reference, and discussed        briefly below:    -   Cong et al. engineered type II CRISPR/Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptoccocus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR/Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Konermann et al. addressed the need in the art for versatile and        robust technologies that enable optical and chemical modulation        of DNA-binding domains based CRISPR Cas9 enzyme and also        Transcriptional Activator Like Effectors    -   Cas9 nuclease from the microbial CRISPR-Cas system is targeted        to specific genomic loci by a 20 nt guide sequence, which can        tolerate certain mismatches to the DNA target and thereby        promote undesired off-target mutagenesis. To address this, Ran        et al. described an approach that combined a Cas9 nickase mutant        with paired guide RNAs to introduce targeted double-strand        breaks. Because individual nicks in the genome are repaired with        high fidelity, simultaneous nicking via appropriately offset        guide RNAs is required for double-stranded breaks and extends        the number of specifically recognized bases for target cleavage.        The authors demonstrated that using paired nicking can reduce        off-target activity by 50- to 1,500-fold in cell lines and to        facilitate gene knockout in mouse zygotes without sacrificing        on-target cleavage efficiency. This versatile strategy enables a        wide variety of genome editing applications that require high        specificity.    -   Hsu et al. characterized SpCas9 targeting specificity in human        cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        reported that SpCas9 tolerates mismatches between guide RNA and        target DNA at different positions in a sequence-dependent        manner, sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and sgRNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. described a set of tools for Cas9-mediated genome        editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNA heteroduplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Hsu 2014 is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells, that is in the information, data and        findings of the applications in the lineage of this        specification filed prior to Jun. 5, 2014. The general teachings        of Hsu 2014 do not involve the specific models, animals of the        instant specification.

Mention is also made of Tsai et al, “Dimeric CRISPR RNA-guided FokInucleases for highly specific genome editing,” Nature Biotechnology32(6): 569-77 (2014) which is not believed to be prior art to theinstant invention or application, but which may be considered in thepractice of the instant invention.

In addition, mention is made of concurrently filed PCT applicationPCT/US14/70057, entitled “DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS ANDDISEASES USING PARTICLE DELIVERY COMPONENTS (claiming priority from oneor more or all of U.S. provisional patent applications: 62/054,490,filed Sep. 24, 2014; 62/010,441, filed Jun. 10, 2014; and 61/915,118,61/915,215 and 61/915,148, each filed on Dec. 12, 2013) (“the ParticleDelivery PCT”), incorporated herein by reference, with respect to amethod of preparing an sgRNA-and-Cas9 protein containing particlecomprising admixing a mixture comprising an sgRNA and Cas9 protein (andoptionally HDR template) with a mixture comprising or consistingessentially of or consisting of surfactant, phospholipid, biodegradablepolymer, lipoprotein and alcohol; and particles from such a process. Forexample, wherein Cas9 protein and sgRNA were mixed together at asuitable, e.g., 3:1 to 1:3 or 2:1 to 1:2 or 1:1 molar ratio, at asuitable temperature, e.g., 15-30 C, e.g., 20-25 C, e.g., roomtemperature, for a suitable time, e.g., 15-45, such as 30 minutes,advantageously in sterile, nuclease free buffer, e.g., 1×PBS.Separately, particle components such as or comprising: a surfactant,e.g., cationic lipid, e.g., 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP); phospholipid, e.g., dimyristoylphosphatidylcholine (DMPC);biodegradable polymer, such as an ethylene-glycol polymer or PEG, and alipoprotein, such as a low-density lipoprotein, e.g., cholesterol weredissolved in an alcohol, advantageously a C₁₋₆ alkyl alcohol, such asmethanol, ethanol, isopropanol, e.g., 100% ethanol. The two solutionswere mixed together to form particles containing the Cas9-sgRNAcomplexes. Accordingly, sgRNA may be pre-complexed with the Cas9protein, before formulating the entire complex in a particle.Formulations may be made with a different molar ratio of differentcomponents known to promote delivery of nucleic acids into cells (e.g.1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP:DMPC:PEG:CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. That applicationaccordingly comprehends admixing sgRNA, Cas9 protein and components thatform a particle; as well as particles from such admixing. Aspects of theinstant invention can involve particles; for example, particles using aprocess analogous to that of the Particle Delivery PCT, e.g., byadmixing a mixture comprising sgRNA and/or Cas9 as in the instantinvention and components that form a particle, e.g., as in the ParticleDelivery PCT, to form a particle and particles from such admixing (or,of course, other particles involving sgRNA and/or Cas9 as in the instantinvention).

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving sequence targeting, such as genome perturbation orgene-editing, that relate to the CRISPR-Cas system and componentsthereof. In advantageous embodiments, the Cas enzyme is Cas9, preferablySpCas9 or SaCas9.

An advantage of the present methods is that the CRISPR system avoidsoff-target binding and its resulting side effects. This is achievedusing systems arranged to have a high degree of sequence specificity forthe target DNA.

Recent advances in the development of genome editing technologies basedon programmable nucleases such as zinc finger nucleases, transcriptionactivator like effector nucleases, and CRISPR-Cas9 have significantlyimproved Applicants' ability to make precise changes in the genomes ofeukaryotic cells. Genome editing is already broadening Applicants'ability to elucidate the contribution of genetics to disease byfacilitating the creation of more accurate cellular and animal models ofpathological processes. A particularly tantalizing application ofprogrammable nucleases is the potential to directly correct geneticmutations in affected tissues and cells to treat genetic diseases thatare refractory to traditional therapies. Applicants provide a discussionherein of the current progress towards developing programmablenuclease-based therapies as well as future prospects and challenges.

Of the approximately 25,000 annotated genes in the human genome,mutations in over 3,000 genes have already been linked to diseasephenotypes, and more disease-relevant genetic variations are beinguncovered at a staggeringly rapid pace. Now, due to sharp drops insequencing cost, the completion of the human genome project, and theexponential growth of genome sequencing data from patients, the role ofgenetics in human health has become a major area of focus for research,clinical medicine and the development of targeted therapeutics [Lander,E. S. Nature 470, 187-197 (2011)]. These advances in Applicants'understanding of the genetic basis of disease have improved Applicants'understanding of disease mechanisms and pointed toward potentialtherapeutic strategies. However, despite valid therapeutic hypothesesand strong efforts in drug development, there have only been a limitednumber of successes using small molecules to treat diseases with stronggenetic contributions [Thoene, J. G. Small molecule therapy for geneticdisease, (Cambridge University Press, Cambridge, UK; New York, 2010)].Thus, alternative approaches are needed. Emerging therapeutic strategiesthat are able to modify nucleic acids within disease-affected cells andtissues hold enormous potential for treatment. Monogenic, highlypenetrant diseases, such as severe-combined immunodeficiency (SCID),haemophilia, and certain enzyme deficiencies have been the focus of suchtherapies due to their well-defined genetics and often lack of safe,effective therapeutic alternatives.

Two of the most powerful genetic therapeutic strategies developed thusfar are viral gene therapy, which enables complementation of missinggene function via transgene expression, and RNA interference (RNAi),which mediates targeted repression of defective genes by knockdown ofthe target mRNA (reviewed in Kay, M. A. Nature reviews. Genetics 12,316-328 (2011) and Vaishnaw, A. K., et al. Silence 1, 14 (2010)). Viralgene therapy has been used to successfully treat monogenic recessivedisorders affecting the hematopoietic system, such as SCID andWiskott-Aldrich syndrome, by semi-randomly integrating functional copiesof affected genes into the genome of hematopoietic stem/progenitor cells[Gaspar, H. B., et al. Science translational medicine 3, 97ra79 (2011),Howe, S. J., et al. The Journal of clinical investigation 118, 3143-3150(2008), Aiuti, A., et al. Science 341, 1233151 (2013)]. RNAi has beenused to repress the function of genes implicated in cancer, age relatedmacular degeneration and TTR-amyloidosis among others, to generate atherapeutic effect in clinical trials, trial numbers: NCT00689065,NCT01961921 and NCT00259753. Despite promise and recent success, viralgene therapy and RNAi have limitations that prevent their utility for alarge number of diseases. For example, viral gene therapy may causeinsertional mutagenesis and dysregulated transgene expression [Howe, S.J., et al. The Journal of clinical investigation 118, 3143-3150 (2008)].Alternatively, RNAi can only repress the expression of target genes,therefore limiting its use to targets where knockdown is beneficial.Also, RNAi often cannot fully repress gene expression, and is thereforeunlikely to provide a benefit for diseases where complete ablation ofgene function is necessary for therapy. An exciting alternative thatmight overcome these limitations would be precise modification of thegenomes of target cells, resulting in the removal or correction ofdeleterious mutations or the insertion of protective mutations. Cartier.

Watts, “Hematopoietic Stem Cell Expansion and Gene Therapy” Cytotherapy13(10):1164-1171. doi:10.3109/14653249.2011.620748 (2011), incorporatedherein by reference along with the documents it cites, as if set out infull, discusses hematopoietic stem cell (HSC) gene therapy, e.g.,virus-mediated hematopoetic stem cell (HSC) gene thereapy, as an highlyattractive treatment option for many disorders including hematologicconditions, immunodeficiencies including HIV/AIDS, and other geneticdisorders like lysosomal storage diseases, including SCID-X1, ADA-SCID,β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia,adrenoleukodystrophy (ALD), and metachromatic leukodystrophy (MLD).

Williams, “Broadening the Indications for Hematopoietic Stem CellGenetic Therapies,” Cell Stem Cell 13:263-264 (2013), incorporatedherein by reference along with the documents it cites, as if set out infull, report lentivirus-mediated gene transfer into HSC/P cells frompatients with the lysosomal storage disease metachromatic leukodystrophydisease (MLD), a genetic disease caused by deficiency of arylsulfatase A(ARSA), resulting in nerve demyelination; and lentivirus-mediated genetransfer into HSCs of patients with Wiskott-Aldrich syndrome (WAS)(patients with defective WAS protein, an effector of the small GTPaseCDC42 that regulates cytoskeletal function in blood cell lineages andthus suffer from immune deficiency with recurrent infections, autoimmunesymptoms, and thrombocytopenia with abnormally small and dysfunctionalplatelets leading to excessive bleeding and an increased risk ofleukemia and lymphoma). In contrast to using lentivirus, with theknowledge in the art and the teachings in this disclosure, the skilledperson can correct HSCs as to MLD (deficiency of arylsulfatase A (ARSA))using a CRISPR-Cas9 system that targets and corrects the mutation(deficiency of arylsulfatase A (ARSA)) (e.g., with a suitable HDRtemplate that delivers a coding sequence for ARSA). In contrast to usinglentivirus, with the knowledge in the art and the teachings in thisdisclosure, the skilled person can correct HSCs as to WAS using aCRISPR-Cas 9 system that targets and corrects the mutation (deficiencyof WAS protein) (e.g., with a suitable HDR template that delivers acoding sequence for WAS protein); specifically, the sgRNA can targetmutation that gives rise to WAS (deficient WAS protein), and the HDR canprovide coding for proper expression of WAS protein.

With the knowledge in the art and the teachings in this disclosure theskilled person can correct HSCs as to immunodeficiency condition such asHIV/AIDS comprising contacting an HSC with a CRISPR-Cas9 system thattargets and knocks out CCR5. An sgRNA (and advantageously a dual guideapproach, e.g., a pair of different sgRNAs; for instance, sgRNAstargeting of two clinically relevant genes, B2M and CCR5, in primaryhuman CD4+ T cells and CD34+ hematopoietic stem and progenitor cells(HSPCs)) that targets and knocks out CCR5- and -Cas9 protein can beintroduced into HSCs. The cells can be administered; and optionallytreated/expanded; cf. Cartier. See also Kiem, “Hematopoietic stemcell-based gene therapy for HIV disease,” Cell Stem Cell. Feb. 3, 2012;10(2): 137-147; incorporated herein by reference along with thedocuments it cites; Mandal et al, “Efficient Ablation of Genes in HumanHematopoietic Stem and Effector Cells using CRISPR/Cas9,” Cell StemCell, Volume 15, Issue 5, p 643-652, 6 Nov. 2014; incorporated herein byreference along with the documents it cites. Mention is also made ofEbina, “CRISPR/Cas9 system to suppress HIV-1 expression by editing HIV-1integrated proviral DNA” SCIENTIFIC REPORTS|3: 2510|DOI:10.1038/srep02510, incorporated herein by reference along with thedocuments it cites, as another means for combatting HIV/AIDS using aCRISPR-Cas9 system.

Genome editing technologies based on programmable nucleases such as zincfinger nuclease (reviewed in Urnov, F. D., et al. Nature reviews.Genetics 11, 636-646 (2010)), transcription activator-like effectornucleases (reviewed in Bogdanove, A. J. & Voytas, D. F. Science 333,1843-1846 (2011)), and clustered regularly interspaced short palindromicrepeat (CRISPR)-associated nuclease Cas9 (reviewed in Hsu, P. D., et al.Cell 157, 1262-1278 (2014)) are opening the possibility of achievingtherapeutic genome editing in diseased cells and tissues. Applicantsprovide a recent review herein.

Genome Editing Technologies

Programmable nucleases enable precise genome editing by introducingtargeted DNA double strand breaks (DSBs) at specific genomic loci. DSBssubsequently signal DNA damage and recruit endogenous repair machineryfor either non-homologous end-joining (NHEJ) or homology directed repair(HDR) to the DSB site to mediate genome editing.

To date, three major classes of nucleases, zinc finger nucleases (ZFNs,FIG. 12, left panel) [Kim, Y. G., et al. Proceedings of the NationalAcademy of Sciences of the United States of America 93, 1156-1160(1996); Wolfe, S. A., et al. Annual review of biophysics andbiomolecular structure 29, 183-212 (2000); Bibikova, M., et al. Science300, 764 (2003); Bibikova, M., et al. Genetics 161, 1169-1175 (2002);Miller, J., et al. The EMBO journal 4, 1609-1614 (1985); Miller, J. C.,et al. Nature biotechnology 25, 778-785 (2007)], transcription activatorlike effector nucleases (TALENs, FIG. 1 middle panel) [Boch, J., et al.Science 326, 1509-1512 (2009); Moscou, M. J. & Bogdanove, A. J. Science326, 1501 (2009); Christian, M., et al. Genetics 186, 757-761 (2010);Miller, J. C., et al. Nature biotechnology 29, 143-148 (2011)], and theCRISPR-associated nuclease Cas9 (FIG. 1, right panel) [Bolotin, A., etal. Microbiology 151, 2551-2561 (2005); Barrangou, R., et al. Science315, 1709-1712 (2007); Garneau, J. E., et al. Nature 468, 67-71 (2010);Deltcheva, E., et al. Nature 471, 602-607 (2011); Sapranauskas, R., etal. Nucleic acids research 39, 9275-9282 (2011); Jinek, M., et al.Science 337, 816-821 (2012); Gasiunas, G., et al. Proceedings of theNational Academy of Sciences of the United States of America 109,E2579-2586 (2012); Cong, L., et al. Science 339, 819-823 (2013); Mali,P., et al. Science 339, 823-826 (2013)], have been developed to enablesite-specific genome editing. These three types of nuclease systems canbe broadly classified into two categories based on their mode of DNArecognition—whereas ZFN and TALEN achieve specific DNA binding viaprotein-DNA interactions, Cas9 is targeted to specific DNA sequences viaa short RNA guide molecule that base-pairs directly with the target DNA(FIG. 13). ZFNs and TALENs are chimeric enzymes consisting of a DNAbinding domain fused to the sequence agnostic nuclease domain, FokI[Kim, Y. G., et al. Proceedings of the National Academy of Sciences ofthe United States of America 93, 1156-1160 (1996); Christian, M., et al.Genetics 186, 757-761 (2010)]. Re-targeting ZFNs and TALENs requireprotein engineering of the DNA binding domain, which is particularlychallenging for ZFNs and still difficult for TALENs [Isalan, M. Naturemethods 9, 32-34 (2012); Sun, N. & Zhao, H. Biotechnology andbioengineering 110, 1811-1821 (2013)]. In contrast, the Cas9 protein isinvariant and can be easily retargeted to new genomic loci by changing asmall portion of the sequence of an accompanying RNA guide. All threenucleases have been demonstrated to achieve efficient genome editing ina wide range of model organisms and mammalian cells and efforts are nowunderway in both industry and academia to develop these tools astherapeutics [Tebas, P., et al. The New England journal of medicine 370,901-910 (2014); Genovese, P., et al. Nature 510, 235-240 (2014); Li, H.,et al. Nature 475, 217-221 (2011); Yin, H., et al. Nature biotechnology32, 551-553 (2014)].

Once the DSB has been made, the lesion may be repaired by either NHEJ orHDR depending on the cell state and the presence of a repair template.NHEJ may repair the lesion by directly rejoining the two DSB ends in aprocess that does not require a repair template. Although NHEJ-mediatedDSB repair can be accurate, repeated repair of the same DSB by NHEJmachinery due to nuclease activity eventually results in the formationof small insertion or deletion mutations bridging the break site[Bibikova, M., et al. Genetics 161, 1169-1175 (2002)]. Such insertionsor deletions (indels) that are introduced into the coding sequence of agene can cause a frameshift mutations that lead to mRNA degradation vianonsense-mediated decay to deplete the functional gene, or results inthe production of nonfunctional truncated proteins [Hentze, M. W. &Kulozik, A. E. Cell 96, 307-310 (1999)]. Thus, NHEJ may be used tosuppress gene function similar to RNAi, however, it leads to continuedsuppression of gene expression in targeted cells by introducingpermanent covalent modifications to the genome.

In comparison, HDR allows researchers to use an exogenous DNA templateto specify the outcome of the DSB repair [Bibikova, M., et al. Science300, 764 (2003); Choulika, A., et al. Molecular and cellular biology 15,1968-1973 (1995); Bibikova, M., et al. Molecular and cellular biology21, 289-297 (2001); Krejci, L., et al. Nucleic acids research 40,5795-5818 (2012); Plessis, A., et al. Genetics 130, 451-460 (1992);Rouet, P., et al. Molecular and cellular biology 14, 8096-8106 (1994);Rudin, N., et al. Genetics 122, 519-534 (1989)]. Upon introduction of atargeted DSB, HDR machinery may use exogenously provided single ordouble stranded DNA templates with sequence homology to the break siteto synthesize DNA that is used to repair the lesion, in the processincorporating any changes encoded in the template DNA. For example, HDRmay be used along with an appropriately designed repair template tocorrect a deleterious mutation directly, thereby restoring gene functionwhile preserving physiological regulation of gene expression.

Considerations for Therapeutic Applications

The first consideration in genome editing therapy is the choice ofsequence-specific nuclease. Each nuclease platform possesses its ownunique set of strengths and weaknesses, many of which must be balancedin the context of treatment to maximize therapeutic benefit (FIG. 12).

Thus far, two therapeutic editing approaches with nucleases have shownsignificant promise: gene disruption and gene correction. Genedisruption involves stimulation of NHEJ to create targeted indels ingenetic elements, often resulting in loss of function mutations that arebeneficial to patients (FIG. 13A). In contrast, gene correction uses HDRto directly reverse a disease causing mutation, restoring function whilepreserving physiological regulation of the corrected element (FIG. 13B).HDR may also be used to insert a therapeutic transgene into a defined‘safe harbor’ locus in the genome to recover missing gene function (FIG.13C).

For a specific editing therapy to be efficacious, a sufficiently highlevel of modification must be achieved in target cell populations toreverse disease symptoms. This therapeutic modification ‘threshold’ isdetermined by the fitness of edited cells following treatment and theamount of gene product necessary to reverse symptoms.

Cell Fitness and Outcome

With regard to fitness, editing creates three potential outcomes fortreated cells relative to their unedited counterparts: increased,neutral, or decreased fitness. In the case of increased fitness, forexample in the treatment of SCID-X1, modified hematopoietic progenitorcells selectively expand relative to their unedited counterparts.SCID-X1 is a disease caused by mutations in the IL2RG gene, the functionof which is required for proper development of the hematopoieticlymphocyte lineage [Leonard, W. J., et al. Immunological reviews 138,61-86 (1994); Kaushansky, K. & Williams, W. J. Williams hematology,(McGraw-Hill Medical, New York, 2010)]. In clinical trials with patientswho received viral gene therapy for SCID-X1, and a rare example of aspontaneous correction of SCID-X1 mutation, corrected hematopoieticprogenitor cells were able to overcome this developmental block andexpand relative to their diseased counterparts to mediate therapy[Bousso, P., et al. Proceedings of the National Academy of Sciences ofthe United States of America 97, 274-278 (2000); Hacein-Bey-Abina, S.,et al. The New England journal of medicine 346, 1185-1193 (2002);Gaspar, H. B., et al. Lancet 364, 2181-2187 (2004)]. In this case, whereedited cells possess a selective advantage, even low numbers of editedcells can be amplified through expansion, providing a therapeuticbenefit to the patient. In contrast, editing for other hematopoieticdiseases, like chronic granulomatous disorder (CGD), would induce nochange in fitness for edited hematopoietic progenitor cells, increasingthe therapeutic modification threshold. CGD is caused by mutations ingenes encoding phagocytic oxidase proteins, which are normally used byneutrophils to generate reactive oxygen species that kill pathogens[Mukherjee, S. & Thrasher, A. J. Gene 525, 174-181 (2013)]. Asdysfunction of these genes does not influence hematopoietic progenitorcell fitness or development, but only the ability of a maturehematopoietic cell type to fight infections, there would be likely nopreferential expansion of edited cells in this disease. Indeed, noselective advantage for gene corrected cells in CGD has been observed ingene therapy trials, leading to difficulties with long-term cellengraftment [Malech, H. L., et al. Proceedings of the National Academyof Sciences of the United States of America 94, 12133-12138 (1997);Kang, H. J., et al. Molecular therapy: the journal of the AmericanSociety of Gene Therapy 19, 2092-2101 (2011)]. As such, significantlyhigher levels of editing would be required to treat diseases like CGD,where editing creates a neutral fitness advantage, relative to diseaseswhere editing creates increased fitness for target cells. If editingimposes a fitness disadvantage, as would be the case for restoringfunction to a tumor suppressor gene in cancer cells, modified cellswould be outcompeted by their diseased counterparts, causing the benefitof treatment to be low relative to editing rates. This latter class ofdiseases would be particularly difficult to treat with genome editingtherapy. X-linked Chronic granulomatous disease (CGD) is a hereditarydisorder of host defense due to absent or decreased activity ofphagocyte NADPH oxidase. From this disclosure and knowledge in the art,the skilled person is enabled to use a CRISPR-Cas9 system that targetsand corrects the mutation (absent or decreased activity of phagocyteNADPH oxidase) (e.g., with a suitable HDR template that delivers acoding sequence for phagocyte NADPH oxidase); specifically, the sgRNAcan target mutation that gives rise to CGD (deficient phagocyte NADPHoxidase), and the HDR can provide coding for proper expression ofphagocyte NADPH oxidase.

In addition to cell fitness, the amount of gene product necessary totreat disease also influences the minimal level of therapeutic genomeediting that must be achieved to reverse symptoms. Haemophilia B is onedisease where a small change in gene product levels can result insignificant changes in clinical outcomes. This disease is caused bymutations in the gene encoding factor IX, a protein normally secreted bythe liver into the blood, where it functions as a component of theclotting cascade. Clinical severity of haemophilia B is related to theamount of factor IX activity. Whereas severe disease is associated withless than 1% of normal activity, milder forms of the diseases areassociated with greater than 1% of factor IX activity [Kaushansky, K. &Williams, W. J. Williams hematology, (McGraw-Hill Medical, New York,2010); Lofqvist, T., et al. Journal of internal medicine 241, 395-400(1997)]. This suggests that editing therapies that can restore factor IXexpression to even a small percentage of liver cells could have a largeimpact on clinical outcomes. A study using ZFNs to correct a mouse modelof haemophilia B shortly after birth demonstrated that 3-7% correctionwas sufficient to reverse disease symptoms, providing preclinicalevidence for this hypothesis [Li, H., et al. Nature 475, 217-221(2011)].

Disorders where a small change in gene product levels can influenceclinical outcomes and diseases where there is a fitness advantage foredited cells, are ideal targets for genome editing therapy, as thetherapeutic modification threshold is low enough to permit a high chanceof success given the current technology.

Targeting these diseases has now resulted in successes with editingtherapy at the preclinical level and a phase I clinical trial (see Tablebelow). Improvements in DSB repair pathway manipulation and nucleasedelivery are needed to extend these promising results to diseases with aneutral fitness advantage for edited cells, or where larger amounts ofgene product are needed for treatment. The Table below shows examples ofapplications of genome editing to therapeutic models.

Nuclease Platform Disease Type Employed Therapeutic Strategy ReferencesHemophilia B ZFN HDR-mediated insertion of correct Li, H., et al. Naturegene sequence 475, 217-221 (2011) HIV ZFN and CRISPR NHEJ-mediatedinactivation of CCR5 Tebas, P., et al. The New England journal ofmedicine 370, 901-910 (2014), Holt, N., et al. Nature biotechnology 28,839-847 (2010), Perez, E. E., et al. Nature biotechnology 26, 808-816(2008), Ye, L., et al. Proceedings of the National Academy of Sciencesof the United States of America 111, 9591-9596 (2014) DMD CRISPR andTALEN NHEJ-mediated removal of stop Ousterout, D. G., et al. codon, andHDR-mediated gene Molecular therapy: the correction journal of theAmerican Society of Gene Therapy 21, 1718-1726 (2013), Long, C., et al.Science 345, 1184-1188 (2014) HBV TALEN and CRISPR NHEJ-mediateddepletion of viral Bloom, K., et al. DNA Molecular therapy: the journalof the American Society of Gene Therapy 21, 1889-1897 (2013), Lin, S.R., et al. Nucleic acids 3, e186 (2014) SCID ZFN HDR-mediated insertionof correct Genovese, P., et al. gene sequence Nature 510, 235-240 (2014)Cataract CRISPR HDR-mediated correction of mutation Wu, Y., et al. Cellstem in mouse zygote cell 13, 659-662 (2013) Cystic fibrosis CRISPRHDR-mediated correction of CFTR in Schwank, G., et al. Cell intestinalstem cell organoid stem cell 13, 653-658 (2013) Hereditary tyrosinemiaCRISPR HDR-mediated correction of mutation Yin, H., et al. Nature inliver biotechnology 32, 551-553 (2014)

An embodiment comprehends contacting a Hemophilia B, SCID (e.g.,SCID-X1, ADA-SCID) or Hereditary tyrosinemia mutation-carryinghematopoetic stem cell with an sgRNA and Cas9 protein targeting agenomic locus of interest as to Hemophilia B, SCID (e.g., SCID-X1,ADA-SCID) or Hereditary tyrosinemia (e.g., as in Li, Genovese or Yin);advantageously with a suitable HDR template to correct the mutation.

Efficiency of DSB Repair Pathways

The activity of NHEJ and HDR DSB repair varies significantly by celltype and cell state. NHEJ is not highly regulated by the cell cycle andis efficient across cell types, allowing for high levels of genedisruption in accessible target cell populations. In contrast, HDR actsprimarily during S/G2 phase, and is therefore restricted to cells thatare actively dividing, limiting treatments that require precise genomemodifications to mitotic cells [Ciccia, A. & Elledge, S. J. Molecularcell 40, 179-204 (2010); Chapman, J. R., et al. Molecular cell 47,497-510 (2012)].

The efficiency of correction via HDR may be controlled by the epigeneticstate or sequence of the targeted locus, or the specific repair templateconfiguration (single vs. double stranded, long vs. short homology arms)used [Hacein-Bey-Abina, S., et al. The New England journal of medicine346, 1185-1193 (2002); Gaspar, H. B., et al. Lancet 364, 2181-2187(2004); Beumer, K. J., et al. G3 (2013)]. The relative activity of NHEJand HDR machineries in target cells may also affect gene correctionefficiency, as these pathways may compete to resolve DSBs [Beumer, K.J., et al. Proceedings of the National Academy of Sciences of the UnitedStates of America 105, 19821-19826 (2008)]. HDR also imposes a deliverychallenge not seen with NHEJ strategies, as it requires the concurrentdelivery of nucleases and repair templates. In practice, theseconstraints have so far led to low levels of HDR in therapeuticallyrelevant cell types. Clinical translation has therefore largely focusedon NHEJ strategies to treat disease, although proof-of-conceptpreclinical HDR treatments have now been described for mouse models ofhaemophilia B and hereditary tyrosinemia [Li, H., et al. Nature 475,217-221 (2011); Yin, H., et al. Nature biotechnology 32, 551-553(2014)].

Cell and Tissue Targeting

Any given genome editing application may comprise combinations ofproteins, small RNA molecules, and/or repair templates, making deliveryof these multiple parts substantially more challenging than smallmolecule therapeutics. Two main strategies for delivery of genomeediting tools have been developed: ex vivo and in vivo. In ex vivotreatments, diseased cells are removed from the body, edited and thentransplanted back into the patient (FIG. 14, top panel). Ex vivo editinghas the advantage of allowing the target cell population to be welldefined and the specific dosage of therapeutic molecules delivered tocells to be specified. The latter consideration may be particularlyimportant when off-target modifications are a concern, as titrating theamount of nuclease may decrease such mutations (Hsu et al., 2013).Another advantage of ex vivo approaches is the typically high editingrates that can be achieved, due to the development of efficient deliverysystems for proteins and nucleic acids into cells in culture forresearch and gene therapy applications.

However, there are two large drawbacks with ex vivo approaches thatlimit their application to a small number of diseases. First, targetcells must be capable of surviving manipulation outside the body. Formany tissues, like the brain, culturing cells outside the body is amajor challenge, because cells either fail to survive, or loseproperties necessary for their function in vivo. Thus, ex vivo therapyis largely limited to tissues with adult stem cell populations amenableto ex vivo culture and manipulation, such as the hematopoietic system.Second, cultured cells often engraft poorly upon re-introduction into apatient, decreasing the effectiveness of treatment. However, engraftmentmay be enhanced by ablative conditioning regimens that deplete hostcells prior to transplantation, which are clinically feasible butintroduce significant risks to patients [Bunn, H. F. & Aster, J.Pathophysiology of blood disorders, (McGraw-Hill, New York, 2011)]

In vivo genome editing involves direct delivery of editing systems tocell types in their native tissues (FIG. 14, bottom panels). In vivoediting allows diseases in which the affected cell population is notamenable to ex vivo manipulation to be treated. Furthermore, deliveringnucleases to cells in situ allows for the treatment of multiple tissueand cell types. These properties probably allow in vivo treatment to beapplied to a wider range of diseases than ex vivo therapies.

To date, in vivo editing has largely been achieved through the use ofviral vectors with defined, tissue-specific tropism. Such vectors arecurrently limited in terms of cargo carrying capacity and tropism,restricting this mode of therapy to organ systems where transductionwith clinically useful vectors is efficient, such as the liver, muscleand eye [Kotterman, M. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Nguyen, T. H. & Ferry, N. Gene therapy 11 Suppl 1,S76-84 (2004); Boye, S. E., et al. Molecular therapy: the journal of theAmerican Society of Gene Therapy 21, 509-519 (2013)].

A major potential barrier for in vivo delivery is the immune responsethat may be created in response to the large amounts of virus necessaryfor treatment, but this phenomenon is not unique to genome editing andis observed with other virus based gene therapies [Bessis, N., et al.Gene therapy 11 Suppl 1, S10-17 (2004)]. It is also possible thatpeptides from editing nucleases themselves are presented on MHC Class Imolecules to stimulate an immune response, although there is littleevidence to support this happening at the preclinical level. Anothermajor difficulty with this mode of therapy is controlling thedistribution and consequently the dosage of genome editing nucleases invivo, leading to off-target mutation profiles that may be difficult topredict.

Examples of Successful Genome Editing Therapeutic Strategies

Ex Vivo Editing Therapy

The long standing clinical expertise with the purification, culture andtransplantation of hematopoietic cells has made diseases affecting theblood system such as SCID, Fanconi anemia, Wiskott-Aldrich syndrome andsickle cell anemia the focus of ex vivo editing therapy. Another reasonto focus on hematopoietic cells is that, thanks to previous efforts todesign gene therapy for blood disorders, delivery systems of relativelyhigh efficiency already exist. Despite these advantages, the often lowefficiency of cell engraftment upon transplantation necessitates thatthis mode of therapy be applied to diseases where edited cells possess afitness advantage, so that a small number of engrafted, edited cells canexpand and treat disease.

Fanconi anemia: Mutations in at least 15 genes (FANCA, FANCB, FANCC,FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BACH1/BRIP1,FANCL/PHF9/POG, FANCM, FANCN/PALB2, FANCO/Rad51C, and FANCP/SLX4/BTBD12)can cause Fanconi anemia. Proteins produced from these genes areinvolved in a cell process known as the FA pathway. The FA pathway isturned on (activated) when the process of making new copies of DNA,called DNA replication, is blocked due to DNA damage. The FA pathwaysends certain proteins to the area of damage, which trigger DNA repairso DNA replication can continue. The FA pathway is particularlyresponsive to a certain type of DNA damage known as interstrandcross-links (ICLs). ICLs occur when two DNA building blocks(nucleotides) on opposite strands of DNA are abnormally attached orlinked together, which stops the process of DNA replication. ICLs can becaused by a buildup of toxic substances produced in the body or bytreatment with certain cancer therapy drugs. Eight proteins associatedwith Fanconi anemia group together to form a complex known as the FAcore complex. The FA core complex activates two proteins, called FANCD2and FANCI. The activation of these two proteins brings DNA repairproteins to the area of the ICL so the cross-link can be removed and DNAreplication can continue. the FA core complex. More in particular, theFA core complex is a nuclear multiprotein complex consisting of FANCA,FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, functions as an E3ubiquitin ligase and mediates the activation of the ID complex, which isa heterodimer composed of FANCD2 and FANCI. Once monoubiquitinated, itinteracts with classical tumor suppressors downstream of the FA pathwayincluding FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1, and FANCO/Rad51C andthereby contributes to DNA repair via homologous recombination (HR).Eighty to 90 percent of FA cases are due to mutations in one of threegenes, FANCA, FANCC, and FANCG. These genes provide instructions forproducing components of the FA core complex. Mutations in such genesassociated with the FA core complex will cause the complex to benonfunctional and disrupt the entire FA pathway. As a result, DNA damageis not repaired efficiently and ICLs build up over time. Geiselhart,“Review Article, Disrupted Signaling through the Fanconi Anemia PathwayLeads to Dysfunctional Hematopoietic Stem Cell Biology: UnderlyingMechanisms and Potential Therapeutic Strategies,” Anemia Volume 2012(2012), Article ID 265790, discussed FA and an animal experimentinvolving intrafemoral injection of a lentivirus encoding the FANCC generesulting in correction of HSCs in vivo. From this disclosure and theknowledge in the art, one can use a CRISPR-Cas9 system that targets andone or more of the mutations associated with FA, for instance aCRISPR-Cas9 system having sgRNA(s) and HDR template(s) that respectivelytargets one or more of the mutations of FANCA, FANCC, or FANCG that giverise to FA and provide corrective expression of one or more of FANCA,FANCC or FANCG.

One such disease is HIV, where infection results in a fitnessdisadvantage to CD4+ T cells.

The rationale for genome editing for HIV treatment originates from theobservation that individuals homozygous for loss of function mutationsin CCR5, a cellular co-receptor for the virus, are highly resistant toinfection and otherwise healthy, suggesting that mimicking this mutationwith genome editing could be a safe and effective therapeutic strategy[Liu, R., et al. Cell 86, 367-377 (1996)]. This idea was clinicallyvalidated when an HIV infected patient was given an allogeneic bonemarrow transplant from a donor homozygous for a loss of function CCR5mutation, resulting in undetectable levels of HIV and restoration ofnormal CD4 T-cell counts [Hutter, G., et al. The New England journal ofmedicine 360, 692-698 (2009)]. Although bone marrow transplantation isnot a realistic treatment strategy for most HIV patients, due to costand potential graft vs. host disease, HIV therapies that convert apatient's own T-cells are realistic.

Early studies using ZFNs and NHEJ to knockout CCR5 in humanized mousemodels of HIV showed that transplantation of CCR5 edited CD4 T cellsimproved viral load and CD4 T-cell counts [Perez, E. E., et al. Naturebiotechnology 26, 808-816 (2008)]. Importantly, these models also showedthat HIV infection resulted in selection for CCR5 null cells, suggestingthat editing confers a fitness advantage and potentially allowing asmall number of edited cells to create a therapeutic effect.

As a result of this and other promising preclinical studies, genomeediting therapy that knocks out CCR5 in patient T cells has now beentested in humans [Holt, N., et al. Nature biotechnology 28, 839-847(2010); Li, L., et al. Molecular therapy: the journal of the AmericanSociety of Gene Therapy 21, 1259-1269 (2013)]. In a recent phase Iclinical trial, CD4+ T cells from patients with HIV were removed, editedwith ZFNs designed to knockout the CCR5 gene, and autologouslytransplanted back into patients [Tebas, P., et al. The New Englandjournal of medicine 370, 901-910 (2014)]. Early results from this trialsuggest that genome editing through ZFNs of the CCR5 locus is safe,although the follow up time is too short to fully understand the risksand efficacy of treatment.

Ex vivo editing therapy has been recently extended to include genecorrection strategies. The barriers to HDR ex vivo were overcome in arecent paper from Genovese and colleagues, who achieved gene correctionof a mutated IL2RG gene in hematopoietic stem cells (HSCs) obtained froma patient suffering from SCID-X1 [Genovese, P., et al. Nature 510,235-240 (2014)]. Genovese et. al. accomplished gene correction in HSCsusing a multimodal strategy. First, HSCs were transduced usingintegration-deficient lentivirus containing an HDR template encoding atherapeutic cDNA for IL2RG. Following transduction, cells wereelectroporated with mRNA encoding ZFNs targeting a mutational hotspot inIL2RG to stimulate HDR based gene correction. To increase HDR rates,culture conditions were optimized with small molecules to encourage HSCdivision. With optimized culture conditions, nucleases and HDRtemplates, gene corrected HSCs from the SCID-X1 patient were obtained inculture at therapeutically relevant rates. HSCs from unaffectedindividuals that underwent the same gene correction procedure couldsustain long-term hematopoiesis in mice, the gold standard for HSCfunction. HSCs are capable of giving rise to all hematopoietic celltypes and can be autologously transplanted, making them an extremelyvaluable cell population for all hematopoietic genetic disorders[Weissman, I. L. & Shizuru, J. A. Blood 112, 3543-3553 (2008)]. Genecorrected HSCs could, in principle, be used to treat a wide range ofgenetic blood disorders making this study an exciting breakthrough fortherapeutic genome editing.

In Vivo Editing Therapy

In vivo editing therapy faces similar challenges to ex vivo strategiesand is also limited by the small number of efficient delivery systems.Inefficient modification of target loci are compounded by anyinefficiencies in delivery, making tissues lacking robust deliveryplatforms particularly difficult to treat with this mode of therapy. Fororgan systems where delivery is efficient, however, there have alreadybeen a number of exciting preclinical therapeutic successes.

The first example of successful in vivo editing therapy was demonstratedin a mouse model of haemophilia B [Li, H., et al. Nature 475, 217-221(2011)]. As noted earlier, Haemophilia B is an X-linked recessivedisorder caused by loss-of-function mutations in the gene encodingFactor IX, a crucial component of the clotting cascade. RecoveringFactor IX activity to above 1% of its levels in severely affectedindividuals can transform the disease into a significantly milder form,as infusion of recombinant Factor IX into such patients prophylacticallyfrom a young age to achieve such levels largely ameliorates clinicalcomplications [Lofqvist, T., et al. Journal of internal medicine 241,395-400 (1997)]. Thus, only low levels of HDR gene correction would benecessary to change clinical outcomes for patients. In addition, FactorIX is synthesized and secreted by the liver, an organ that can betransduced efficiently by viral vectors encoding editing systems. Withthe knowledge in the art and the teachings in this disclosure, theskilled person can correct HSCs as to Haemophilia B using a CRISPR-Cas9system that targets and corrects the mutation (X-linked recessivedisorder caused by loss-of-function mutations in the gene encodingFactor IX) (e.g., with a suitable HDR template that delivers a codingsequence for Factor IX); specifically, the sgRNA can target mutationthat give rise to Haemophilia B, and the HDR can provide coding forproper expression of Factor IX.

Using hepatotropic adeno-associated viral (AAV) serotypes encoding ZFNsand a corrective HDR template, up to 7% gene correction of a mutated,humanized Factor IX gene in the murine liver was achieved [Li, H., etal. Nature 475, 217-221 (2011)]. This resulted in improvement of clotformation kinetics, a measure of the function of the clotting cascade,demonstrating for the first time that in vivo editing therapy is notonly feasible, but also efficacious.

Building on this study, other groups have recently used in vivo genomeediting of the liver with CRISPR-Cas9 to successfully treat a mousemodel of hereditary tyrosinemia and to create mutations that provideprotection against cardiovascular disease. These two distinctapplications demonstrate the versatility of this approach for disordersthat involve hepatic dysfunction [Yin, H., et al. Nature biotechnology32, 551-553 (2014); Ding, Q., et al. Circulation research 115, 488-492(2014)]. Application of in vivo editing to other organ systems arenecessary to prove that this strategy is widely applicable. Currently,efforts to optimize both viral and non-viral vectors are underway toexpand the range of disorders that can be treated with this mode oftherapy [Kotterman, M. A. & Schaffer, D. V. Nature reviews. Genetics 15,445-451 (2014); Yin, H., et al. Nature reviews. Genetics 15, 541-555(2014)].

Specificity of Editing Nucleases

The specificity of genome editing tools is one of the main safetyconcerns for clinical application. Genetic modifications are permanent,and deleterious off-target mutations have the potential to create cellswith oncogenic potential and other undesirable side effects.Furthermore, oncogenic mutations resulting from off-target editing maylead to expansion of the edited the cells, thus, even low levels ofoff-target mutagenesis may have devastating consequences.

Two issues remain outstanding: evaluating and reducing off-targeteffects. A number of studies have attempted to evaluate the targetingspecificity of ZFN, TALEN, and Cas9 nucleases. The limited number ofstudies characterizing ZFN [Pattanayak, V., et al. Nature methods 8,765-770 (2011); Gabriel, R., et al. Nature biotechnology 29, 816-823(2011)] and TALEN [Guilinger, J. P., et al. Nature methods 11, 429-435(2014)] specificity have only highlighted the challenges of detectingZFN and TALEN off-target activity. Of note, the two independent studiesattempting to characterize the off-target profile of the same pair ofCCR5-targeting ZFNs have returned distinct and non-overlappingoff-target sites, which highlights the challenges associated withanalysis of nuclease specificity.

Many studies have attempted to evaluate the specificity of Cas9, partlyowing to the simplicity of the RNA-guided DNA targeting mechanism ofCas9, which makes it significantly easier to establish hypothesesregarding possible off-targeting mechanisms based on Watson-Crick basepairing rules. While initial bacterial [Sapranauskas, R., et al. Nucleicacids research 39, 9275-9282 (2011)], biochemical [Jinek, M., et al.Science 337, 816-821 (2012); Gasiunas, G., et al. Proceedings of theNational Academy of Sciences of the United States of America 109,E2579-2586 (2012)], and mammalian [Cong, L., et al. Science 339, 819-823(2013)] experiments have suggested that the 3′ 8-12 bp seed region ofthe guide sequence can be sensitive to single base mismatches, furtherwork have shown that this rule-of-thumb is not necessarily accurate,especially in situations where there is high concentration of Cas9 andguide RNA [Fu, Y., et al. Nature biotechnology 31, 822-826 (2013); Cho,S. W., et al. Genome research 24, 132-141 (2014); Hsu, P. D., et al.Nature biotechnology 31, 827-832 (2013); Mali, P., et al. Naturebiotechnology 31, 833-838 (2013); Pattanayak, V., et al. Naturebiotechnology 31, 839-843 (2013)]. Many of these studies were carriedout in cell lines and examined Cas9-mediated mutagenesis at genomicsites bearing high levels of homology to the on-target sequence andfound that, unsurprisingly, subsets of highly homologous off-targetsites were significantly mutated by the nuclease. However, the scope ofpossible off-target sites evaluated by these studies was limited tocomputationally predicted sites. More recently, whole-genome sequencingof Cas9-edited cell lines revealed low incidence of off-target mutation,which suggests that Cas9-mediated genome editing can be specific [Veres,A., et al. Cell stem cell 15, 27-30 (2014)]. Despite these studies,unbiased assessment of genome wide off-targeting using more advancedmethods like direct capture of DSBs [Crosetto, N., et al. Nature methods10, 361-365 (2013)] and techniques that can detect larger structuralvariations (i.e. translocations) potentially imposed by nucleasetreatment remains an urgent need and should be undertaken to understandthe true risk of mutagenesis imposed by programmable nucleases. It isworth noting that off-target effects may be cell-type specific; forexample off-target effects in transformed cell lines with dysergulatedDSB repair pathways may overestimate off-target effects that would beobserved in primary healthy cells.

In order to reduce the frequency of off-target effects, many groups arerapidly improving the targeting specificity of Cas9. For example,transformation of Cas9 into a single-strand DNA nickase that functionsas an obligate heterodimer dramatically reduces off-target indelformation at computationally predicted off target sites [Mali, P., etal. Nature biotechnology 31, 833-838 (2013); Ran, F. A., et al. Cell154, 1380-1389 (2013)]. Additionally, truncation of the guide RNA aswell as RNA-guided FokI nuclease based on fusion between catalyticallyinactive Cas9 and the FokI nuclease domain are also able to achieveimproved levels of targeting specificity [Fu, Y., et al. Naturebiotechnology 32, 279-284 (2014); Guilinger, J. P., et al. Naturebiotechnology 32, 577-582 (2014); Tsai, S. Q., et al. Naturebiotechnology 32, 569-576 (2014)]. These, and future, improved nucleasestrategies are considered, whenever possible, for therapeuticapplications.

Crispr-Cas Systems and Compositions for Therapeutic Applications, e.g.,Genome Editing

In general, in addition to discussion throughout this documentconcerning the CRISPR-Cas or CRISPR system, the CRISPR-Cas or CRISPRsystem is as used in the herein-cited documents, such as WO 2014/093622(PCT/US2013/074667) and refers collectively to transcripts and otherelements involved in the expression of or directing the activity ofCRISPR-associated (“Cas”) genes, including sequences encoding a Casgene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or anactive partial tracrRNA), a tracr-mate sequence (encompassing a “directrepeat” and a tracrRNA-processed partial direct repeat in the context ofan endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or “RNA(s)” asthat term is herein used (e.g., RNA(s) to guide Cas9, e.g. CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). In the context of formation of a CRISPR complex, “targetsequence” refers to a sequence to which a guide sequence is designed tohave complementarity, where hybridization between a target sequence anda guide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, direct repeatsmay be identified in silico by searching for repetitive motifs thatfulfill any or all of the following criteria: 1. found in a 2 Kb windowof genomic sequence flanking the type II CRISPR locus; 2. span from 20to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 ofthese criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3.In some embodiments, all 3 criteria may be used. In some embodiments itmay be preferred in a CRISPR complex that the tracr sequence has one ormore hairpins and is 30 or more nucleotides in length, 40 or morenucleotides in length, or 50 or more nucleotides in length; the guidesequence is between 10 to 30 nucleotides in length, the CRISPR/Casenzyme is a Type II Cas9 enzyme. In embodiments of the invention theterms guide sequence and guide RNA are used interchangeably as inforegoing cited documents such as WO 2014/093622 (PCT/US2013/074667). Ingeneral, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. In some embodiments, thedegree of complementarity between a guide sequence and its correspondingtarget sequence, when optimally aligned using a suitable alignmentalgorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 99%, or more. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences, non-limitingexample of which include the Smith-Waterman algorithm, theNeedleman-Wunsch algorithm, algorithms based on the Burrows-WheelerTransform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT,Novoalign (Novocraft Technologies; available at www novocraft.com),ELAND (Illumina, San Diego, Calif.), SOAP (available atsoap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10-30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art. A guide sequence may be selected to target any target sequence.In some embodiments, the target sequence is a sequence within a genomeof a cell. Exemplary target sequences include those that are unique inthe target genome. For example, for the S. pyogenes Cas9, a uniquetarget sequence in a genome may include a Cas9 target site of the formMMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO: 4) where NNNNNNNNNNNNXGG (SEQ ID NO:5) (N is A, G, T, or C; and X can be anything) has a single occurrencein the genome. A unique target sequence in a genome may include an S.pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG (SEQ IDNO: 6) where NNNNNNNNNNNXGG (SEQ ID NO: 7) (N is A, G, T, or C; and Xcan be anything) has a single occurrence in the genome. For the S.thermophilus CRISPR1 Cas9, a unique target sequence in a genome mayinclude a Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQID NO: 8) where NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 9) (N is A, G, T, or C;X can be anything; and W is A or T) has a single occurrence in thegenome. A unique target sequence in a genome may include an S.thermophilus CRISPR1 Cas9 target site of the formMMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 10) where NNNNNNNNNNNXXAGAAW(SEQ ID NO: 11) (N is A, G, T, or C; X can be anything; and W is A or T)has a single occurrence in the genome. For the S. pyogenes Cas9, aunique target sequence in a genome may include a Cas9 target site of theform MMMMMMMMNNNNNNNNNNNNXGGXG (SEQ ID NO: 12) where NNNNNNNNNNNNXGGXG(SEQ ID NO: 13) (N is A, G, T, or C; and X can be anything) has a singleoccurrence in the genome. A unique target sequence in a genome mayinclude an S. pyogenes Cas9 target site of the formMMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 14) where NNNNNNNNNNNXGGXG (SEQ IDNO: 15) (N is A, G, T, or C; and X can be anything) has a singleoccurrence in the genome. In each of these sequences “M” may be A, G, T,or C, and need not be considered in identifying a sequence as unique. Insome embodiments, a guide sequence is selected to reduce the degreesecondary structure within the guide sequence. In some embodiments,about or less than about 75%, 50%, 40% a, 30%, 25%, 20%, 15%, 10%, 5%,1%, or fewer of the nucleotides of the guide sequence participate inself-complementary base pairing when optimally folded. Optimal foldingmay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and P A Carr and G M Church, 2009, Nature Biotechnology27(12): 1151-62).

In general, a tracr mate sequence includes any sequence that hassufficient complementarity with a tracr sequence to promote one or moreof: (1) excision of a guide sequence flanked by tracr mate sequences ina cell containing the corresponding tracr sequence; and (2) formation ofa CRISPR complex at a target sequence, wherein the CRISPR complexcomprises the tracr mate sequence hybridized to the tracr sequence. Ingeneral, degree of complementarity is with reference to the optimalalignment of the tracr mate sequence and tracr sequence, along thelength of the shorter of the two sequences. Optimal alignment may bedetermined by any suitable alignment algorithm, and may further accountfor secondary structures, such as self-complementarity within either thetracr sequence or tracr mate sequence. In some embodiments, the degreeof complementarity between the tracr sequence and tracr mate sequencealong the length of the shorter of the two when optimally aligned isabout or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,97.5%, 99%, or higher. In some embodiments, the tracr sequence is aboutor more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 40, 50, or more nucleotides in length. In someembodiments, the tracr sequence and tracr mate sequence are containedwithin a single transcript, such that hybridization between the twoproduces a transcript having a secondary structure, such as a hairpin.In an embodiment of the invention, the transcript or transcribedpolynucleotide sequence has at least two or more hairpins. In preferredembodiments, the transcript has two, three, four or five hairpins. In afurther embodiment of the invention, the transcript has at most fivehairpins. In a hairpin structure the portion of the sequence 5′ of thefinal “N” and upstream of the loop corresponds to the tracr matesequence, and the portion of the sequence 3′ of the loop corresponds tothe tracr sequence Further non-limiting examples of singlepolynucleotides comprising a guide sequence, a tracr mate sequence, anda tracr sequence are as follows (listed 5′ to 3′), where “N” representsa base of a guide sequence, the first block of lower case lettersrepresent the tracr mate sequence, and the second block of lower caseletters represent the tracr sequence, and the final poly-T sequencerepresents the transcription terminator: (1)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ IDNO: 16); (2)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 17);(3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 18); (4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 19); (5)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT (SEQ ID NO: 20); and (6)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT (SEQ ID NO: 21). In some embodiments, sequences (1) to (3) areused in combination with Cas9 from S. thermophilus CRISPR1. In someembodiments, sequences (4) to (6) are used in combination with Cas9 fromS. pyogenes. In some embodiments, the tracr sequence is a separatetranscript from a transcript comprising the tracr mate sequence.

In some embodiments, candidate tracrRNA may be subsequently predicted bysequences that fulfill any or all of the following criteria: 1. sequencehomology to direct repeats (motif search in Geneious with up to 18-bpmismatches); 2. presence of a predicted Rho-independent transcriptionalterminator in direction of transcription; and 3. stable hairpinsecondary structure between tracrRNA and direct repeat. In someembodiments, 2 of these criteria may be used, for instance 1 and 2, 2and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

In some embodiments, chimeric synthetic guide RNAs (sgRNAs) designs mayincorporate at least 12 bp of duplex structure between the direct repeatand tracrRNA.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of CRISPR enzyme mRNA and guide RNAdelivered. Optimal concentrations of CRISPR enzyme mRNA and guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing the analyzethe extent of modification at potential off-target genomic loci. Forexample, for the guide sequence targeting 5′-GAGTCCGAGCAGAAGAAGAA-3′(SEQ ID NO: 22) in the EMX1 gene of the human genome, deep sequencingcan be used to assess the level of modification at the following twooff-target loci, 1: 5′-GAGTCCTAGCAGGAGAAGAA-3′ (SEQ ID NO: 23) and 2:5′-GAGTCTAAGCAGAAGAAGAA-3′ (SEQ ID NO: 24). The concentration that givesthe highest level of on-target modification while minimizing the levelof off-target modification should be chosen for in vivo delivery.Alternatively, to minimize the level of toxicity and off-target effect,CRISPR enzyme nickase mRNA (for example S. pyogenes Cas9 with the D10Amutation) can be delivered with a pair of guide RNAs targeting a site ofinterest. The two guide RNAs need to be spaced as follows. Guidesequences and strategies to minimize toxicity and off-target effects canbe as in WO 2014/093622 (PCT/US2013/074667).

The CRISPR system is derived advantageously from a type II CRISPRsystem. In some embodiments, one or more elements of a CRISPR system isderived from a particular organism comprising an endogenous CRISPRsystem, such as Streptococcus pyogenes. In preferred embodiments of theinvention, the CRISPR system is a type II CRISPR system and the Casenzyme is Cas9, which catalyzes DNA cleavage. Non-limiting examples ofCas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7,Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, ormodified versions thereof.

In some embodiments, the unmodified CRISPR enzyme has DNA cleavageactivity, such as Cas9. In some embodiments, the CRISPR enzyme directscleavage of one or both strands at the location of a target sequence,such as within the target sequence and/or within the complement of thetarget sequence. In some embodiments, the CRISPR enzyme directs cleavageof one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 50, 100, 200, 500, or more base pairs from the first or lastnucleotide of a target sequence. In some embodiments, a vector encodes aCRISPR enzyme that is mutated to with respect to a correspondingwild-type enzyme such that the mutated CRISPR enzyme lacks the abilityto cleave one or both strands of a target polynucleotide containing atarget sequence. For example, an aspartate-to-alanine substitution(D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes convertsCas9 from a nuclease that cleaves both strands to a nickase (cleaves asingle strand). Other examples of mutations that render Cas9 a nickaseinclude, without limitation, H840A, N854A, and N863A. As a furtherexample, two or more catalytic domains of Cas9 (RuvC I, RuvC II, andRuvC III or the HNH domain) may be mutated to produce a mutated Cas9substantially lacking all DNA cleavage activity. In some embodiments, aD10A mutation is combined with one or more of H840A, N854A, or N863Amutations to produce a Cas9 enzyme substantially lacking all DNAcleavage activity. In some embodiments, a CRISPR enzyme is considered tosubstantially lack all DNA cleavage activity when the DNA cleavageactivity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%,0.1%, 0.01%, or less of the DNA cleavage activity of the non-mutatedform of the enzyme; an example can be when the DNA cleavage activity ofthe mutated form is nil or negligible as compared with the non-mutatedform. Where the enzyme is not SpCas9, mutations may be made at any orall residues corresponding to positions 10, 762, 840, 854, 863 and/or986 of SpCas9 (which may be ascertained for instance by standardsequence comparison tools). In particular, any or all of the followingmutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863Aand/or D986A; as well as conservative substitution for any of thereplacement amino acids is also envisaged. The same (or conservativesubstitutions of these mutations) at corresponding positions in otherCas9s are also preferred. Particularly preferred are D10 and H840 inSpCas9. However, in other Cas9s, residues corresponding to SpCas9 D10and H840 are also preferred. For instance in Sa Cas9, a mutation atN580, e.g., N580A is advantageous. Orthologs of SpCas9 can be used inthe practice of the invention. A Cas enzyme may be identified Cas9 asthis can refer to the general class of enzymes that share homology tothe biggest nuclease with multiple nuclease domains from the type IICRISPR system. Most preferably, the Cas9 enzyme is from, or is derivedfrom, spCas9 (S. pyogenes Cas9) or saCas9 (S. aureus Cas9). StCas9”refers to wild type Cas9 from S. thermophilus, the protein sequence ofwhich is given in the SwissProt database under accession number G3ECR1.Similarly, S. pyogenes Cas9 or spCas9 is included in SwissProt underaccession number Q99ZW2. By derived, Applicants mean that the derivedenzyme is largely based, in the sense of having a high degree ofsequence homology with, a wildtype enzyme, but that it has been mutated(modified) in some way as described herein. It will be appreciated thatthe terms Cas and CRISPR enzyme are generally used hereininterchangeably, unless otherwise apparent. As mentioned above, many ofthe residue numberings used herein refer to the Cas9 enzyme from thetype II CRISPR locus in Sreptococcus pyogenes. However, it will beappreciated that this invention includes many more Cas9s from otherspecies of microbes, such as SpCas9, SaCa9, St1Cas9 and so forth.Enzymatic action by Cas9 derived from Streptococcus pyogenes or anyclosely related Cas9 generates double stranded breaks at target sitesequences which hybridize to 20 nucleotides of the guide sequence andthat have a protospacer-adjacent motif (PAM) sequence (examples includeNGG/NRG or a PAM that can be determined as described herein) followingthe 20 nucleotides of the target sequence. CRISPR activity through Cas9for site-specific DNA recognition and cleavage is defined by the guidesequence, the tracr sequence that hybridizes in part to the guidesequence and the PAM sequence. More aspects of the CRISPR system aredescribed in Karginov and Hannon, The CRISPR system: small RNA-guideddefence in bacteria and archaea, Mole Cell 2010, Jan. 15; 37(1): 7. Thetype II CRISPR locus from Streptococcus pyogenes SF370, which contains acluster of four genes Cas9, Cas1, Cas2, and Csn1, as well as twonon-coding RNA elements, tracrRNA and a characteristic array ofrepetitive sequences (direct repeats) interspaced by short stretches ofnon-repetitive sequences (spacers, about 30 bp each). In this system,targeted DNA double-strand break (DSB) is generated in four sequentialsteps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, aretranscribed from the CRISPR locus. Second, tracrRNA hybridizes to thedirect repeats of pre-crRNA, which is then processed into mature crRNAscontaining individual spacer sequences. Third, the mature crRNA:tracrRNAcomplex directs Cas9 to the DNA target consisting of the protospacer andthe corresponding PAM via heteroduplex formation between the spacerregion of the crRNA and the protospacer DNA. Finally, Cas9 mediatescleavage of target DNA upstream of PAM to create a DSB within theprotospacer. A pre-crRNA array consisting of a single spacer flanked bytwo direct repeats (DRs) is also encompassed by the term “tracr-matesequences”). In certain embodiments, Cas9 may be constitutively presentor inducibly present or conditionally present or administered ordelivered. Cas9 optimization may be used to enhance function or todevelop new functions, one can generate chimeric Cas9 proteins. And Cas9may be used as a generic DNA binding protein.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.Without wishing to be bound by theory, the tracr sequence, which maycomprise or consist of all or a portion of a wild-type tracr sequence(e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, ormore nucleotides of a wild-type tracr sequence), may also form part of aCRISPR complex, such as by hybridization along at least a portion of thetracr sequence to all or a portion of a tracr mate sequence that isoperably linked to the guide sequence.

An example of a codon optimized sequence, is in this instance a sequenceoptimized for expression in a eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667). Whilst this ispreferred, it will be appreciated that other examples are possible andcodon optimization for a host species other than human, or for codonoptimization for specific organs is known. In some embodiments, anenzyme coding sequence encoding a CRISPR enzyme is codon optimized forexpression in particular cells, such as eukaryotic cells. The eukaryoticcells may be those of or derived from a particular organism, such as amammal, including but not limited to human, or non-human eukaryote oranimal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog,livestock, or non-human mammal or primate. In some embodiments,processes for modifying the germ line genetic identity of human beingsand/or processes for modifying the genetic identity of animals which arelikely to cause them suffering without any substantial medical benefitto man or animal, and also animals resulting from such processes, may beexcluded. In general, codon optimization refers to a process ofmodifying a nucleic acid sequence for enhanced expression in the hostcells of interest by replacing at least one codon (e.g. about or morethan about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of thenative sequence with codons that are more frequently or most frequentlyused in the genes of that host cell while maintaining the native aminoacid sequence. Various species exhibit particular bias for certaincodons of a particular amino acid. Codon bias (differences in codonusage between organisms) often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, among other things, the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, for example, at the “Codon Usage Database” and thesetables can be adapted in a number of ways. See Nakamura, Y., et al.“Codon usage tabulated from the international DNA sequence databases:status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computeralgorithms for codon optimizing a particular sequence for expression ina particular host cell are also available, such as Gene Forge (Aptagen;Jacobus, Pa.), are also available. In some embodiments, one or morecodons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons)in a sequence encoding a CRISPR enzyme correspond to the most frequentlyused codon for a particular amino acid.

In some embodiments, a vector encodes a CRISPR enzyme comprising one ormore nuclear localization sequences (NLSs), such as about or more thanabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments,the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near thecarboxy-terminus, or a combination of these (e.g. zero or at least oneor more NLS at the amino-terminus and zero or at one or more NLS at thecarboxy terminus). When more than one NLS is present, each may beselected independently of the others, such that a single NLS may bepresent in more than one copy and/or in combination with one or moreother NLSs present in one or more copies. In a preferred embodiment ofthe invention, the CRISPR enzyme comprises at most 6 NLSs. In someembodiments, an NLS is considered near the N- or C-terminus when thenearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20,25, 30, 40, 50, or more amino acids along the polypeptide chain from theN- or C-terminus. Non-limiting examples of NLSs include an NLS sequencederived from: the NLS of the SV40 virus large T-antigen, having theamino acid sequence PKKKRKV (SEQ ID NO: 25); the NLS from nucleoplasmin(e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK(SEQ ID NO: 26)); the c-myc NLS having the amino acid sequence PAAKRVKLD(SEQ ID NO: 27) or RQRRNELKRSP (SEQ ID NO: 28); the hRNPA1 M9 NLS havingthe sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 29); thesequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 30) ofthe IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:31) and PPKKARED (SEQ ID NO: 32) of the myoma T protein; the sequencePQPKKKPL (SEQ ID NO: 33) of human p53: the sequence SALIKKKKKMAP (SEQ IDNO: 34) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 35) andPKQKKRK (SEQ ID NO: 36) of the influenza virus NS1; the sequenceRKLKKKIKKL (SEQ ID NO: 37) of the Hepatitis virus delta antigen; thesequence REKKKFLKRR (SEQ ID NO: 38) of the mouse M×1 protein; thesequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 39) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNO: 40) of the steroid hormone receptors (human) glucocorticoid. Ingeneral, the one or more NLSs are of sufficient strength to driveaccumulation of the CRISPR enzyme in a detectable amount in the nucleusof a eukaryotic cell. In general, strength of nuclear localizationactivity may derive from the number of NLSs in the CRISPR enzyme, theparticular NLS(s) used, or a combination of these factors. Detection ofaccumulation in the nucleus may be performed by any suitable technique.For example, a detectable marker may be fused to the CRISPR enzyme, suchthat location within a cell may be visualized, such as in combinationwith a means for detecting the location of the nucleus (e.g. a stainspecific for the nucleus such as DAPI). Cell nuclei may also be isolatedfrom cells, the contents of which may then be analyzed by any suitableprocess for detecting protein, such as immunohistochemistry, Westernblot, or enzyme activity assay. Accumulation in the nucleus may also bedetermined indirectly, such as by an assay for the effect of CRISPRcomplex formation (e.g. assay for DNA cleavage or mutation at the targetsequence, or assay for altered gene expression activity affected byCRISPR complex formation and/or CRISPR enzyme activity), as compared toa control no exposed to the CRISPR enzyme or complex, or exposed to aCRISPR enzyme lacking the one or more NLSs.

Aspects of the invention relate to the expression of the gene productbeing decreased or a template polynucleotide being further introducedinto the DNA molecule encoding the gene product or an interveningsequence being excised precisely by allowing the two 5′ overhangs toreanneal and ligate or the activity or function of the gene productbeing altered or the expression of the gene product being increased. Inan embodiment of the invention, the gene product is a protein. OnlysgRNA pairs creating 5′ overhangs with less than 8 bp overlap betweenthe guide sequences (offset greater than −8 bp) were able to mediatedetectable indel formation. Importantly, each guide used in these assaysis able to efficiently induce indels when paired with wildtype Cas9,indicating that the relative positions of the guide pairs are the mostimportant parameters in predicting double nicking activity. Since Cas9nand Cas9H840A nick opposite strands of DNA, substitution of Cas9n withCas9H840A with a given sgRNA pair should have resulted in the inversionof the overhang type; but no indel formation is observed as withCas9H840A indicating that Cas9H840A is a CRISPR enzyme substantiallylacking all DNA cleavage activity (which is when the DNA cleavageactivity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%,0.1%, 0.01%, or less of the DNA cleavage activity of the non-mutatedform of the enzyme; whereby an example can be when the DNA cleavageactivity of the mutated form is nil or negligible as compared with thenon-mutated form, e.g., when no indel formation is observed as withCas9H840A in the eukaryotic system in contrast to the biochemical orprokaryotic systems). Nonetheless, a pair of sgRNAs that will generate a5′ overhang with Cas9n should in principle generate the corresponding 3′overhang instead, and double nicking. Therefore, sgRNA pairs that leadto the generation of a 3′ overhang with Cas9n can be used with anothermutated Cas9 to generate a 5′ overhang, and double nicking. Accordingly,in some embodiments, a recombination template is also provided. Arecombination template may be a component of another vector as describedherein, contained in a separate vector, or provided as a separatepolynucleotide. In some embodiments, a recombination template isdesigned to serve as a template in homologous recombination, such aswithin or near a target sequence nicked or cleaved by a CRISPR enzyme asa part of a CRISPR complex. A template polynucleotide may be of anysuitable length, such as about or more than about 10, 15, 20, 25, 50,75, 100, 150, 200, 500, 1000, or more nucleotides in length. In someembodiments, the template polynucleotide is complementary to a portionof a polynucleotide comprising the target sequence. When optimallyaligned, a template polynucleotide might overlap with one or morenucleotides of a target sequences (e.g. about or more than about 1, 5,10, 15, 20, or more nucleotides). In some embodiments, when a templatesequence and a polynucleotide comprising a target sequence are optimallyaligned, the nearest nucleotide of the template polynucleotide is withinabout 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000,10000, or more nucleotides from the target sequence.

In some embodiments, one or more vectors driving expression of one ormore elements of a CRISPR system are introduced into a host cell suchthat expression of the elements of the CRISPR system direct formation ofa CRISPR complex at one or more target sites. For example, a Cas enzyme,a guide sequence linked to a tracr-mate sequence, and a tracr sequencecould each be operably linked to separate regulatory elements onseparate vectors. Or, RNA(s) of the CRISPR System can be delivered to atransgenic Cas9 animal or mammal, e.g., an animal or mammal thatconstitutively or inducibly or conditionally expresses Cas9; or ananimal or mammal that is otherwise expressing Cas9 or has cellscontaining Cas9, such as by way of prior administration thereto of avector or vectors that code for and express in vivo Cas9. Alternatively,two or more of the elements expressed from the same or differentregulatory elements, may be combined in a single vector, with one ormore additional vectors providing any components of the CRISPR systemnot included in the first vector. CRISPR system elements that arecombined in a single vector may be arranged in any suitable orientation,such as one element located 5′ with respect to (“upstream” of) or 3′with respect to (“downstream” of) a second element. The coding sequenceof one element may be located on the same or opposite strand of thecoding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a CRISPR enzyme and one or more ofthe guide sequence, tracr mate sequence (optionally operably linked tothe guide sequence), and a tracr sequence embedded within one or moreintron sequences (e.g. each in a different intron, two or more in atleast one intron, or all in a single intron). In some embodiments, theCRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequenceare operably linked to and expressed from the same promoter. Deliveryvehicles, vectors, particles, nanoparticles, formulations and componentsthereof for expression of one or more elements of a CRISPR system are asused in the foregoing documents, such as WO 2014/093622(PCT/US2013/074667). In some embodiments, a vector comprises one or moreinsertion sites, such as a restriction endonuclease recognition sequence(also referred to as a “cloning site”). In some embodiments, one or moreinsertion sites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more insertion sites) are located upstream and/or downstreamof one or more sequence elements of one or more vectors. In someembodiments, a vector comprises an insertion site upstream of a tracrmate sequence, and optionally downstream of a regulatory elementoperably linked to the tracr mate sequence, such that followinginsertion of a guide sequence into the insertion site and uponexpression the guide sequence directs sequence-specific binding of aCRISPR complex to a target sequence in a eukaryotic cell. In someembodiments, a vector comprises two or more insertion sites, eachinsertion site being located between two tracr mate sequences so as toallow insertion of a guide sequence at each site. In such anarrangement, the two or more guide sequences may comprise two or morecopies of a single guide sequence, two or more different guidesequences, or combinations of these. When multiple different guidesequences are used, a single expression construct may be used to targetCRISPR activity to multiple different, corresponding target sequenceswithin a cell. For example, a single vector may comprise about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guidesequences. In some embodiments, about or more than about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may beprovided, and optionally delivered to a cell. In some embodiments, avector comprises a regulatory element operably linked to anenzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.CRISPR enzyme or CRISPR enzyme mRNA or CRISPR guide RNA or RNA(s) can bedelivered separately; and advantageously at least one of these isdelivered via a nanoparticle complex. CRISPR enzyme mRNA can bedelivered prior to the guide RNA to give time for CRISPR enzyme to beexpressed. CRISPR enzyme mRNA might be administered 1-12 hours(preferably around 2-6 hours) prior to the administration of guide RNA.Alternatively, CRISPR enzyme mRNA and guide RNA can be administeredtogether. Advantageously, a second booster dose of guide RNA can beadministered 1-12 hours (preferably around 2-6 hours) after the initialadministration of CRISPR enzyme mRNA+guide RNA. Additionaladministrations of CRISPR enzyme mRNA and/or guide RNA might be usefulto achieve the most efficient levels of genome modification.

In one aspect, the invention provides methods for using one or moreelements of a CRISPR system. The CRISPR complex of the inventionprovides an effective means for modifying a target polynucleotide. TheCRISPR complex of the invention has a wide variety of utility includingmodifying (e.g., deleting, inserting, translocating, inactivating,activating) a target polynucleotide in a multiplicity of cell types. Assuch the CRISPR complex of the invention has a broad spectrum ofapplications in, e.g., gene therapy, drug screening, disease diagnosis,and prognosis. An exemplary CRISPR complex comprises a CRISPR enzymecomplexed with a guide sequence hybridized to a target sequence withinthe target polynucleotide. The guide sequence is linked to a tracr matesequence, which in turn hybridizes to a tracr sequence. In oneembodiment, this invention provides a method of cleaving a targetpolynucleotide. The method comprises modifying a target polynucleotideusing a CRISPR complex that binds to the target polynucleotide andeffect cleavage of said target polynucleotide. Typically, the CRISPRcomplex of the invention, when introduced into a cell, creates a break(e.g., a single or a double strand break) in the genome sequence. Forexample, the method can be used to cleave a disease gene in a cell. Thebreak created by the CRISPR complex can be repaired by a repairprocesses such as the error prone non-homologous end joining (NHEJ)pathway or the high fidelity homology-directed repair (HDR). Duringthese repair process, an exogenous polynucleotide template can beintroduced into the genome sequence. In some methods, the HDR process isused modify genome sequence. For example, an exogenous polynucleotidetemplate comprising a sequence to be integrated flanked by an upstreamsequence and a downstream sequence is introduced into a cell. Theupstream and downstream sequences share sequence similarity with eitherside of the site of integration in the chromosome. Where desired, adonor polynucleotide can be DNA, e.g., a DNA plasmid, a bacterialartificial chromosome (BAC), a yeast artificial chromosome (YAC), aviral vector, a linear piece of DNA, a PCR fragment, a naked nucleicacid, or a nucleic acid complexed with a delivery vehicle such as aliposome or poloxamer. The exogenous polynucleotide template comprises asequence to be integrated (e.g., a mutated gene). The sequence forintegration may be a sequence endogenous or exogenous to the cell.Examples of a sequence to be integrated include polynucleotides encodinga protein or a non-coding RNA (e.g., a microRNA). Thus, the sequence forintegration may be operably linked to an appropriate control sequence orsequences. Alternatively, the sequence to be integrated may provide aregulatory function. The upstream and downstream sequences in theexogenous polynucleotide template are selected to promote recombinationbetween the chromosomal sequence of interest and the donorpolynucleotide. The upstream sequence is a nucleic acid sequence thatshares sequence similarity with the genome sequence upstream of thetargeted site for integration. Similarly, the downstream sequence is anucleic acid sequence that shares sequence similarity with thechromosomal sequence downstream of the targeted site of integration. Theupstream and downstream sequences in the exogenous polynucleotidetemplate can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identitywith the targeted genome sequence. Preferably, the upstream anddownstream sequences in the exogenous polynucleotide template have about95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targetedgenome sequence. In some methods, the upstream and downstream sequencesin the exogenous polynucleotide template have about 99% or 100% sequenceidentity with the targeted genome sequence. An upstream or downstreamsequence may comprise from about 20 bp to about 2500 bp, for example,about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,or 2500 bp. In some methods, the exemplary upstream or downstreamsequence have about 200 bp to about 2000 bp, about 600 bp to about 1000bp, or more particularly about 700 bp to about 1000 bp. In some methods,the exogenous polynucleotide template may further comprise a marker.Such a marker may make it easy to screen for targeted integrations.Examples of suitable markers include restriction sites, fluorescentproteins, or selectable markers. The exogenous polynucleotide templateof the invention can be constructed using recombinant techniques (see,for example, Sambrook et al., 2001 and Ausubel et al., 1996). In amethod for modifying a target polynucleotide by integrating an exogenouspolynucleotide template, a double stranded break is introduced into thegenome sequence by the CRISPR complex, the break is repaired viahomologous recombination an exogenous polynucleotide template such thatthe template is integrated into the genome. The presence of adouble-stranded break facilitates integration of the template. In otherembodiments, this invention provides a method of modifying expression ofa polynucleotide in a eukaryotic cell. The method comprises increasingor decreasing expression of a target polynucleotide by using a CRISPRcomplex that binds to the polynucleotide. In some methods, a targetpolynucleotide can be inactivated to effect the modification of theexpression in a cell. For example, upon the binding of a CRISPR complexto a target sequence in a cell, the target polynucleotide is inactivatedsuch that the sequence is not transcribed, the coded protein is notproduced, or the sequence does not function as the wild-type sequencedoes. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. In some methods, a control sequence can be inactivatedsuch that it no longer functions as a control sequence. As used herein,“control sequence” refers to any nucleic acid sequence that effects thetranscription, translation, or accessibility of a nucleic acid sequence.Examples of a control sequence include, a promoter, a transcriptionterminator, and an enhancer are control sequences. The targetpolynucleotide of a CRISPR complex can be any polynucleotide endogenousor exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Examples of targetpolynucleotides include a sequence associated with a signalingbiochemical pathway, e.g., a signaling biochemical pathway-associatedgene or polynucleotide. Examples of target polynucleotides include adisease associated gene or polynucleotide. A “disease-associated” geneor polynucleotide refers to any gene or polynucleotide which is yieldingtranscription or translation products at an abnormal level or in anabnormal form in cells derived from a disease-affected tissues comparedwith tissues or cells of a non disease control. It may be a gene thatbecomes expressed at an abnormally high level; it may be a gene thatbecomes expressed at an abnormally low level, where the alteredexpression correlates with the occurrence and/or progression of thedisease. A disease-associated gene also refers to a gene possessingmutation(s) or genetic variation that is directly responsible or is inlinkage disequilibrium with a gene(s) that is responsible for theetiology of a disease. The transcribed or translated products may beknown or unknown, and may be at a normal or abnormal level. The targetpolynucleotide of a CRISPR complex can be any polynucleotide endogenousor exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Without wishing to be bound bytheory, it is believed that the target sequence should be associatedwith a PAM (protospacer adjacent motif); that is, a short sequencerecognized by the CRISPR complex. The precise sequence and lengthrequirements for the PAM differ depending on the CRISPR enzyme used, butPAMs are typically 2-5 base pair sequences adjacent the protospacer(that is, the target sequence) Examples of PAM sequences are given inthe examples section below, and the skilled person will be able toidentify further PAM sequences for use with a given CRISPR enzyme. Insome embodiments, the method comprises allowing a CRISPR complex to bindto the target polynucleotide to effect cleavage of said targetpolynucleotide thereby modifying the target polynucleotide, wherein theCRISPR complex comprises a CRISPR enzyme complexed with a guide sequencehybridized to a target sequence within said target polynucleotide,wherein said guide sequence is linked to a tracr mate sequence which inturn hybridizes to a tracr sequence. In one aspect, the inventionprovides a method of modifying expression of a polynucleotide in aeukaryotic cell. In some embodiments, the method comprises allowing aCRISPR complex to bind to the polynucleotide such that said bindingresults in increased or decreased expression of said polynucleotide;wherein the CRISPR complex comprises a CRISPR enzyme complexed with aguide sequence hybridized to a target sequence within saidpolynucleotide, wherein said guide sequence is linked to a tracr matesequence which in turn hybridizes to a tracr sequence. Similarconsiderations and conditions apply as above for methods of modifying atarget polynucleotide. In fact, these sampling, culturing andre-introduction options apply across the aspects of the presentinvention. In one aspect, the invention provides for methods ofmodifying a target polynucleotide in a eukaryotic cell, which may be invivo, ex vivo or in vitro. In some embodiments, the method comprisessampling a cell or population of cells from a human or non-human animal,and modifying the cell or cells. Culturing may occur at any stage exvivo. The cell or cells may even be re-introduced into the non-humananimal or plant. For re-introduced cells it is particularly preferredthat the cells are stem cells.

Indeed, in any aspect of the invention, the CRISPR complex may comprisea CRISPR enzyme complexed with a guide sequence hybridized to a targetsequence, wherein said guide sequence may be linked to a tracr matesequence which in turn may hybridize to a tracr sequence.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving sequence targeting, such as genome perturbation orgene-editing, that relate to the CRISPR-Cas system and componentsthereof. In advantageous embodiments, the Cas enzyme is Cas9. Anadvantage of the present methods is that the CRISPR system minimizes oravoids off-target binding and its resulting side effects. This isachieved using systems arranged to have a high degree of sequencespecificity for the target DNA.

Self-Inactivating Systems

Once intended alterations have been introduced, such as by editingintended copies of a gene in the genome of a cell, continued CRISRP/Cas9expression in that cell is no longer necessary. Indeed, sustainedexpression would be undesirable in certain casein case of off-targeteffects at unintended genomic sites, etc. Thus time-limited expressionwould be useful. Inducible expression offers one approach, but inaddition Applicants have engineered a Self-Inactivating CRISPR-Cas9system that relies on the use of a non-coding guide target sequencewithin the CRISPR vector itself. Thus, after expression begins, theCRISPR system will lead to its own destruction, but before destructionis complete it will have time to edit the genomic copies of the targetgene (which, with a normal point mutation in a diploid cell, requires atmost two edits). Simply, the self inactivating CRISPR-Cas systemincludes additional RNA (i.e., guide RNA) that targets the codingsequence for the CRISPR enzyme itself or that targets one or morenon-coding guide target sequences complementary to unique sequencespresent in one or more of the following: (a) within the promoter drivingexpression of the non-coding RNA elements, (b) within the promoterdriving expression of the Cas9 gene, (c) within 100 bp of the ATGtranslational start codon in the Cas9 coding sequence, (d) within theinverted terminal repeat (iTR) of a viral delivery vector, e.g., in anAAV genome.

The Efficiency of HDR

Although the amount of genome modification in a target cell populationrequired to create a therapeutic effect differs depending on thedisease, the efficacy of most editing treatments are improved withincreased editing rates. As previously noted, editing rates arecontrolled by the activity of DSB repair pathways and the efficiency ofdelivery to cells of interest. Therefore improvements to either one ofthese factors are likely to improve the efficacy of editing treatments.

Attempts to increase the activity rates of DSB repair pathways havelargely focused on HDR, as cell cycle regulation and the challenge ofdelivering an HDR template with nucleases makes strategies employingthis pathway less efficient than NHEJ. Cell cycle regulation has nowbeen somewhat by-passed for slowly cycling cell types throughstimulation of mitosis with pharmacologic agents ex vivo [Kormann, M.S., et al. Nature biotechnology 29, 154-157 (2011)]. However, trulypost-mitotic cells are unlikely to be amenable to such manipulation,limiting the applicability of this strategy. Attempts have been made tocompletely circumvent the need for HDR through direct ligation of DNAtemplates containing therapeutic transgenes into targeted DSBs. Suchligation events have been observed, but the rates are too low to beuseful for therapy [Ran, F. A., et al. Cell 154, 1380-1389 (2013);Orlando, S. J., et al. Nucleic acids research 38, e152 (2010)]. Likelydramatically new approaches are necessary to improve HDR efficiency andincrease the therapeutic efficacy of strategies requiring precisegenomic correction.

Genome editing presents tantalizing opportunities for tackling a numberof intractable diseases. Nevertheless, the technology is still in itsinfancy and require a number of iterations to systematically optimizeits efficacy, safety, and specificity. Additionally, despite theenormous excitement surrounding genome editing, strategic planning andrigorous but enabling regulatory processes are necessary to ensuresuccessful development of this class of potentially life-changingmedicine.

Delivery, Including Delivery Generally

A variety of nucleic acid or protein delivery methods may be used tointroduce genome editing nucleases into target cells ex vivo or in vivo.Depending on the choice of delivery method, the nucleases may either betransiently or permanently expressed in the target cell. Given thatnucleases may exhibit off-target cleavage activity or trigger immuneresponses, the delivery system should be carefully selected. For ex vivoapplications, such as editing of hematopoietic stem cells,electroporation may be used to achieve transient nuclease expressionthrough delivery of DNA-based nuclease expression vectors, mRNA, orprotein. Both integration-competent and deficient lentiviral vectorshave also been successfully used to drive nuclease expression. However,integrating lentiviral vectors may be less desirable as they driveconstitutive expression and may result in more off-target activity. Inaddition, all three nuclease platform have also been demonstrated to beamenable to modifications so that proteins can be directly deliveredinto cells either through engineered cell-penetrating or chemicalconjugation [Guilinger, J. P., et al. Nature methods 11, 429-435 (2014);Zuris, J. A., et al. Nature biotechnology (2014); Gaj, T., et al. Naturemethods 9, 805-807 (2012)].

For in vivo applications, the most promising delivery systems are viralvectors, particularly adeno-associated viral (AAV) vectors, which haverecently been approved for clinical use [Wirth, T., et al. Gene 525,162-169 (2013)]. AAV comes in many serotypes and have been shown to havehigh delivery efficacy for a variety of tissue types including the eye,brain, liver, and muscle [Samulski, R. J. & Muzyczka, N. Annual Reviewof Virology 1, 427-451 (2014)]. However, the relatively small packagingcapacity of AAV vectors post some challenges for nuclease delivery.Whereas ZFNs are relatively small and a dimeric ZFN pair can be packagedinto a single AAV, a dimeric TALEN pair is much larger and likely needto be packaged into two separate AAV vectors. For Cas9, short orthologsmay be packaged along with guide RNAs into a single AAV. So far,AAV-mediated nuclease expression has been demonstrated to be successfulin several tissue types, including liver and brain [Li, H., et al.Nature 475, 217-221 (2011); Swiech, L., et al. Nature biotechnology(2014)].

Despite the potential of AAV-mediated in vivo nuclease expression, thereare several challenges that require further development. First,AAV-mediated nuclease expression is often constitutive and it would bemore desirable to be able to shut down nuclease expression after thegenome editing event has successfully occurred in the target cell.Second, patients who have already been naturally exposed to AAV likelyhave developed immunity against specific serotypes. Therefore AAV maynot be an appropriate delivery vehicle for these patients. To overcomethese challenges faced by viral vectors, nanoparticle- and lipid-basedin vivo mRNA or protein delivery systems may provide an attractivealternative [Zuris, J. A., et al. Nature biotechnology (2014); Kormann,M. S., et al. Nature biotechnology 29, 154-157 (2011)].

Through this disclosure and the knowledge in the art, CRISPR-Cas system,or components thereof or nucleic acid molecules thereof (including, forinstance HDR template) or nucleic acid molecules encoding or providingcomponents thereof may be delivered by a delivery system hereindescribed both generally and in detail.

Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, forinstance a Cas9, and/or any of the present RNAs, for instance a guideRNA, can be delivered using any suitable vector, e.g., plasmid or viralvectors, such as adeno associated virus (AAV), lentivirus, adenovirus orother viral vector types, or combinations thereof. Cas9 and one or moreguide RNAs can be packaged into one or more vectors, e.g., plasmid orviral vectors. In some embodiments, the vector, e.g., plasmid or viralvector is delivered to the tissue of interest by, for example, anintramuscular injection, while other times the delivery is viaintravenous, transdermal, intranasal, oral, mucosal, or other deliverymethods. Such delivery may be either via a single dose, or multipledoses. One skilled in the art understands that the actual dosage to bedelivered herein may vary greatly depending upon a variety of factors,such as the vector choice, the target cell, organism, or tissue, thegeneral condition of the subject to be treated, the degree oftransformation/modification sought, the administration route, theadministration mode, the type of transformation/modification sought,etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10⁰ particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10⁷ pu, about 2×10⁷ pu, about 4×10⁷ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu, about 2×10¹⁰ pu, about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding a CRISPRenzyme, operably linked to said promoter; (iii) a selectable marker;(iv) an origin of replication; and (v) a transcription terminatordownstream of and operably linked to (ii). The plasmid can also encodethe RNA components of a CRISPR complex, but one or more of these mayinstead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example. Tolentino etal., Retina 24(4):660 which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver Cas9 and gRNA (and, for instance, HR repairtemplate) into cells using liposomes or nanoparticles. Thus delivery ofthe CRISPR enzyme, such as a Cas9 and/or delivery of the RNAs of theinvention may be in RNA form and via microvesicles, liposomes ornanoparticles. For example, Cas9 mRNA and gRNA can be packaged intoliposomal particles for delivery in vivo. Liposomal transfectionreagents such as lipofectamine from Life Technologies and other reagentson the market can effectively deliver RNA molecules into the liver.

Means of delivery of RNA also preferred include delivery of RNA viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe CRISPR system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012Nov. 15.) describe how exosomes are promising tools for drug deliveryacross different biological barriers and can be harnessed for deliveryof siRNA in vitro and in vivo. Their approach is to generate targetedexosomes through transfection of an expression vector, comprising anexosomal protein fused with a peptide ligand. The exosomes are thenpurify and characterized from transfected cell supernatant, then RNA isloaded into the exosomes. Delivery or administration according to theinvention can be performed with exosomes, in particular but not limitedto the brain. Vitamin E (α-tocopherol) may be conjugated with CRISPR Casand delivered to the brain along with high density lipoprotein (HDL),for example in a similar manner as was done by Uno et al. (HUMAN GENETHERAPY 22:711-719 (June 2011)) for delivering short-interfering RNA(siRNA) to the brain. Mice were infused via Osmotic minipumps (model1007D; Alzet, Cupertino, Calif.) filled with phosphate-buffered saline(PBS) or free TocsiBACE or Toc-siBACE/HDL and connected with BrainInfusion Kit 3 (Alzet). A brain-infusion cannula was placed about 0.5 mmposterior to the bregma at midline for infusion into the dorsal thirdventricle. Uno et al. found that as little as 3 nmol of Toc-siRNA withHDL could induce a target reduction in comparable degree by the same ICVinfusion method. A similar dosage of CRISPR Cas conjugated toα-tocopherol and co-administered with HDL targeted to the brain may becontemplated for humans in the present invention, for example, about 3nmol to about 3 pmol of CRISPR Cas targeted to the brain may becontemplated. Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April 2011))describes a method of lentiviral-mediated delivery of short-hairpin RNAstargeting PKCy for in vivo gene silencing in the spinal cord of rats.Zou et al. administered about 10 μl of a recombinant lentivirus having atiter of 1×109 transducing units (TU)/ml by an intrathecal catheter. Asimilar dosage of CRISPR Cas expressed in a lentiviral vector targetedto the brain may be contemplated for humans in the present invention,for example, about 10-50 ml of CRISPR Cas targeted to the brain in alentivirus having a titer of 1×109 transducing units (TU)/ml may becontemplated.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g. byinjection. Injection can be performed stereotactically via a craniotomy.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and animals comprisingor produced from such cells. In some embodiments, a CRISPR enzyme incombination with (and optionally complexed with) a guide sequence isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues.

Such methods can be used to administer nucleic acids encoding componentsof a CRISPR system to cells in culture, or in a host organism. Non-viralvector delivery systems include DNA plasmids, RNA (e.g. a transcript ofa vector described herein), naked nucleic acid, and nucleic acidcomplexed with a delivery vehicle, such as a liposome. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell. For a review of genetherapy procedures, see Anderson, Science 256:808-813 (1992); Nabel &Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada etal., in Current Topics in Microbiology and Immunology Doerfler and Böhm(eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994). Methods ofnon-viral delivery of nucleic acids include lipofection, microinjection,biolistics, virosomes, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection is described in e.g., U.S.Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagentsare sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424; WO91/16024. Delivery can be to cells (e.g. in vitro or ex vivoadministration) or target tissues (e.g. in vivo administration). Thepreparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues. The tropism of a retrovirus canbe altered by incorporating foreign envelope proteins, expanding thepotential target population of target cells. Lentiviral vectors areretroviral vectors that are able to transduce or infect non-dividingcells and typically produce high viral titers. Selection of a retroviralgene transfer system would therefore depend on the target tissue.Retroviral vectors are comprised of cis-acting long terminal repeatswith packaging capacity for up to 6-10 kb of foreign sequence. Theminimum cis-acting LTRs are sufficient for replication and packaging ofthe vectors, which are then used to integrate the therapeutic gene intothe target cell to provide permanent transgene expression. Widely usedretroviral vectors include those based upon murine leukemia virus(MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus(SIV), human immuno deficiency virus (HIV), and combinations thereof(see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann etal., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59(1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J.Virol. 65:2220-2224 (1991); PCT/US94/05700). In another embodiment,Cocal vesiculovirus envelope pseudotyped retroviral vector particles arecontemplated (see, e.g., US Patent Publication No. 20120164118 assignedto the Fred Hutchinson Cancer Research Center). Cocal virus is in theVesiculovirus genus, and is a causative agent of vesicular stomatitis inmammals. Cocal virus was originally isolated from mites in Trinidad(Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)), and infectionshave been identified in Trinidad, Brazil, and Argentina from insects,cattle, and horses. Many of the vesiculoviruses that infect mammals havebeen isolated from naturally infected arthropods, suggesting that theyare vector-borne. Antibodies to vesiculoviruses are common among peopleliving in rural areas where the viruses are endemic andlaboratory-acquired; infections in humans usually result ininfluenza-like symptoms. The Cocal virus envelope glycoprotein shares71.5% identity at the amino acid level with VSV-G Indiana, andphylogenetic comparison of the envelope gene of vesiculoviruses showsthat Cocal virus is serologically distinct from, but most closelyrelated to, VSV-G Indiana strains among the vesiculoviruses. Jonkers etal., Am. J. Vet. Res. 25:236-242 (1964) and Travassos da Rosa et al.,Am. J. Tropical Med. & Hygiene 33:999-1006 (1984). The Cocalvesiculovirus envelope pseudotyped retroviral vector particles mayinclude for example, lentiviral, alpharetroviral, betaretroviral,gammaretroviral, deltaretroviral, and epsilonretroviral vector particlesthat may comprise retroviral Gag, Pol, and/or one or more accessoryprotein(s) and a Cocal vesiculovirus envelope protein. Within certainaspects of these embodiments, the Gag, Pol, and accessory proteins arelentiviral and/or gammaretroviral. In applications where transientexpression is preferred, adenoviral based systems may be used.Adenoviral based vectors are capable of very high transductionefficiency in many cell types and do not require cell division. Withsuch vectors, high titer and levels of expression have been obtained.This vector can be produced in large quantities in a relatively simplesystem. Adeno-associated virus (“AAV”) vectors may also be used totransduce cells with target nucleic acids, e.g., in the in vitroproduction of nucleic acids and peptides, and for in vivo and ex vivogene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368: WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989). Packaging cells aretypically used to form virus particles that are capable of infecting ahost cell. Such cells include 293 cells, which package adenovirus, andψ2 cells or PA317 cells, which package retrovirus. Viral vectors used ingene therapy are usually generated by producer a cell line that packagesa nucleic acid vector into a viral particle. The vectors typicallycontain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the polynucleotide(s) to be expressed. Themissing viral functions are typically supplied in trans by the packagingcell line. For example, AAV vectors used in gene therapy typically onlypossess ITR sequences from the AAV genome which are required forpackaging and integration into the host genome. Viral DNA is packaged ina cell line, which contains a helper plasmid encoding the other AAVgenes, namely rep and cap, but lacking ITR sequences. The cell line mayalso be infected with adenovirus as a helper. The helper virus promotesreplication of the AAV vector and expression of AAV genes from thehelper plasmid. The helper plasmid is not packaged in significantamounts due to a lack of ITR sequences. Contamination with adenoviruscan be reduced by, e.g., heat treatment to which adenovirus is moresensitive than AAV. Accordingly, AAV is considered an ideal candidatefor use as a transducing vector. Such AAV transducing vectors cancomprise sufficient cis-acting functions to replicate in the presence ofadenovirus or herpesvirus or poxvirus (e.g., vaccinia virus) helperfunctions provided in trans. Recombinant AAV (rAAV) can be used to carryexogenous genes into cells of a variety of lineages. In these vectors,the AAV cap and/or rep genes are deleted from the viral genome andreplaced with a DNA segment of choice. Current AAV vectors mayaccommodate up to 4300 bases of inserted DNA. There are a number of waysto produce rAAV, and the invention provides rAAV and methods forpreparing rAAV. For example, plasmid(s) containing or consistingessentially of the desired viral construct are transfected intoAAV-infected cells. In addition, a second or additional helper plasmidis cotransfected into these cells to provide the AAV rep and/or capgenes which are obligatory for replication and packaging of therecombinant viral construct. Under these conditions, the rep and/or capproteins of AAV act in trans to stimulate replication and packaging ofthe rAAV construct. Two to Three days after transfection, rAAV isharvested. Traditionally rAAV is harvested from the cells along withadenovirus. The contaminating adenovirus is then inactivated by heattreatment. In the instant invention, rAAV is advantageously harvestednot from the cells themselves, but from cell supernatant. Accordingly,in an initial aspect the invention provides for preparing rAAV, and inaddition to the foregoing, rAAV can be prepared by a method thatcomprises or consists essentially of: infecting susceptible cells with arAAV containing exogenous DNA including DNA for expression, and helpervirus (e.g., adenovirus, herpesvirus, poxvirus such as vaccinia virus)wherein the rAAV lacks functioning cap and/or rep (and the helper virus(e.g., adenovirus, herpesvirus, poxvirus such as vaccinia virus)provides the cap and/or rev function that the rAAV lacks); or infectingsusceptible cells with a rAAV containing exogenous DNA including DNA forexpression, wherein the recombinant lacks functioning cap and/or rep,and transfecting said cells with a plasmid supplying cap and/or repfunction that the rAAV lacks; or infecting susceptible cells with a rAAVcontaining exogenous DNA including DNA for expression, wherein therecombinant lacks functioning cap and/or rep, wherein said cells supplycap and/or rep function that the recombinant lacks; or transfecting thesusceptible cells with an AAV lacking functioning cap and/or rep andplasmids for inserting exogenous DNA into the recombinant so that theexogenous DNA is expressed by the recombinant and for supplying repand/or cap functions whereby transfection results in an rAAV containingthe exogenous DNA including DNA for expression that lacks functioningcap and/or rep. The rAAV can be from an AAV as herein described, andadvantageously can be an rAAV1, rAAV2, AAV5 or rAAV having hybrid orcapsid which may comprise AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the rAAV with regard to the cells to betargeted by the rAAV; e.g., one can select AAV serotypes 1, 2, 5 or ahybrid or capsid AAV1, AAV2, AAV5 or any combination thereof fortargeting brain or neuronal cells; and one can select AAV4 for targetingcardiac tissue. In addition to 293 cells, other cells that can be usedin the practice of the invention and the relative infectivity of certainAAV serotypes in vitro as to these cells (see Grimm, D. et al, J. Virol.82: 5887-5911 (2008)) are as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.00.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 NDND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND333 3333 ND ND

The invention provides rAAV that contains or consists essentially of anexogenous nucleic acid molecule encoding a CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeats) system, e.g., a plurality ofcassettes comprising or consisting a first cassette comprising orconsisting essentially of a promoter, a nucleic acid molecule encoding aCRISPR-associated (Cas) protein (putative nuclease or helicaseproteins), e.g., Cas9 and a terminator, and a two, or more,advantageously up to the packaging size limit of the vector, e.g., intotal (including the first cassette) five, cassettes comprising orconsisting essentially of a promoter, nucleic acid molecule encodingguide RNA (gRNA) and a terminator (e.g., each cassette schematicallyrepresented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . .. Promoter-gRNA(N)-terminator (where N is a number that can be insertedthat is at an upper limit of the packaging size limit of the vector), ortwo or more individual rAAVs, each containing one or more than onecassette of a CRISPR system, e.g., a first rAAV containing the firstcassette comprising or consisting essentially of a promoter, a nucleicacid molecule encoding Cas, e.g., Cas9 and a terminator, and a secondrAAV containing a plurality, four, cassettes comprising or consistingessentially of a promoter, nucleic acid molecule encoding guide RNA(gRNA) and a terminator (e.g., each cassette schematically representedas Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . . .Promoter-gRNA(N)-terminator (where N is a number that can be insertedthat is at an upper limit of the packaging size limit of the vector). AsrAAV is a DNA virus, the nucleic acid molecules in the herein discussionconcerning AAV or rAAV are advantageously DNA. The promoter is in someembodiments advantageously human Synapsin I promoter (hSyn). Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference. In some embodiments, a host cell is transiently ornon-transiently transfected with one or more vectors described herein.In some embodiments, a cell is transfected as it naturally occurs in asubject. In some embodiments, a cell that is transfected is taken from asubject. In some embodiments, the cell is derived from cells taken froma subject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr−/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a CRISPR system as described herein (such as by transienttransfection of one or more vectors, or transfection with RNA), andmodified through the activity of a CRISPR complex, is used to establisha new cell line comprising cells containing the modification but lackingany other exogenous sequence. In some embodiments, cells transiently ornon-transiently transfected with one or more vectors described herein,or cell lines derived from such cells are used in assessing one or moretest compounds.

Enhancing NHEJ or HR efficiency is also helpful for delivery. It ispreferred that NHEJ efficiency is enhanced by co-expressingend-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011August; 188(4): 787-797). It is preferred that HR efficiency isincreased by transiently inhibiting NHEJ machineries such as Ku70 andKu86. HR efficiency can also be increased by co-expressing prokaryoticor eukaryotic homologous recombination enzymes such as RecBCD, RecA.

Packaging and Promoters Generally

Ways to package Cas9 coding nucleic acid molecules, e.g., DNA, intovectors, e.g., viral vectors, to mediate genome modification in vivoinclude:

To achieve NHEJ-mediated gene knockout:

-   -   Single virus vector:    -   Vector containing two or more expression cassettes:    -   Promoter-Cas9 coding nucleic acid molecule-terminator    -   Promoter-gRNA1-terminator    -   Promoter-gRNA2-terminator    -   Promoter-gRNA(N)-terminator (up to size limit of vector)

Double virus vector:

-   -   Vector 1 containing one expression cassette for driving the        expression of Cas9    -   Promoter-Cas9 coding nucleic acid molecule-terminator    -   Vector 2 containing one more expression cassettes for driving        the expression of one or more guideRNAs    -   Promoter-gRNA1-terminator    -   Promoter-gRNA(N)-terminator (up to size limit of vector)

To mediate homology-directed repair.

-   -   In addition to the single and double virus vector approaches        described above, an additional vector is used to deliver a        homology-direct repair template.

The promoter used to drive Cas9 coding nucleic acid molecule expressioncan include:

-   -   AAV ITR can serve as a promoter: this is advantageous for        eliminating the need for an additional promoter element (which        can take up space in the vector). The additional space freed up        can be used to drive the expression of additional elements        (gRNA, etc.). Also, ITR activity is relatively weaker, so can be        used to reduce potential toxicity due to over expression of        Cas9.    -   For ubiquitous expression, can use promoters: CMV, CAG, CBh,        PGK, SV40, Ferritin heavy or light chains, etc.

For brain or other CNS expression, can use promoters: SynapsinI for allneurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT forGABAergic neurons, etc.

For liver expression, can use Albumin promoter.

For lung expression, can use SP-B.

For endothelial cells, can use ICAM.

For hematopoietic cells can use IFNbeta or CD45.

For Osteoblasts can use OG-2.

The promoter used to drive guide RNA can include:

Pol III promoters such as U6 or H1

Use of Pol II promoter and intronic cassettes to express gRNA

Adeno Associated Virus (AAV)

Cas9 and one or more guide RNA can be delivered using adeno associatedvirus (AAV), lentivirus, adenovirus or other plasmid or viral vectortypes, in particular, using formulations and doses from, for example,U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat.No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946(formulations, doses for DNA plasmids) and from clinical trials andpublications regarding the clinical trials involving lentivirus, AAV andadenovirus. For examples, for AAV, the route of administration,formulation and dose can be as in U.S. Pat. No. 8,454,972 and as inclinical trials involving AAV. For Adenovirus, the route ofadministration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g. a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specific genomemodification, the expression of Cas9 can be driven by a cell-typespecific promoter. For example, liver-specific expression might use theAlbumin promoter and neuron-specific expression (e.g. for targeting CNSdisorders) might use the Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultra centrifugation of cell particles that can        activate the immune response)    -   Low probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cas9 aswell as a promoter and transcription terminator have to be all fit intothe same viral vector. Constructs larger than 4.5 or 4.75 Kb will leadto significantly reduced virus production. SpCas9 is quite large, thegene itself is over 4.1 Kb, which makes it difficult for packing intoAAV. Therefore embodiments of the invention include utilizing homologsof Cas9 that are shorter. For example:

Species Cas9 Size Corynebacter diphtheriae 3252 Eubacterium ventriosum3321 Streptococcus pasteurianus 3390 Lactobacillus farciminis 3378Sphaerochaeta globus 3537 Azospirillum B510 3504 Gluconacetobacterdiazotrophicus 3150 Neisseria cinerea 3246 Roseburia intestinalis 3420Parvibaculum lavamentivorans 3111 Staphylococcus aureus 3159Nitratifractor salsuginis DSM 16511 3396 Campylobacter lari CF89-12 3009Streptococcus thermophilus LMD-9 3396

These species are therefore, in general, preferred Cas9 species.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al, J. Virol. 82:5887-5911 (2008)) is as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.00.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 NDND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND333 3333 ND ND

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCasES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to OptiMEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 μg of lentiviral transfer plasmid (pCasES10) and the followingpackaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 ug ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with acationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4 C. They were then aliquottedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinfectious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the CRISPR-Cas system of the presentinvention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the CRISPR-Cas system of the presentinvention. A minimum of 2.5×106 CD34+ cells per kilogram patient weightmay be collected and prestimulated for 16 to 20 hours in X-VIVO 15medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×106 cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm2 tissue culture flasks coated with fibronectin (25mg/cm2) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

RNA Delivery

RNA delivery: The CRISPR enzyme, for instance a Cas9, and/or any of thepresent RNAs, for instance a guide RNA, can also be delivered in theform of RNA. Cas9 mRNA can be generated using in vitro transcription.For example, Cas9 mRNA can be synthesized using a PCR cassettecontaining the following elements: T7_promoter-kozak sequence(GCCACC)-Cas9-3′ UTR from beta globin-polyA tail (a string of 120 ormore adenines (SEQ ID NO: 193)). The cassette can be used fortranscription by T7 polymerase. Guide RNAs can also be transcribed usingin vitro transcription from a cassette containing T7_promoter-GG-guideRNA sequence.

To enhance expression and reduce possible toxicity, the CRISPRenzyme-coding sequence and/or the guide RNA can be modified to includeone or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.

mRNA delivery methods are especially promising for liver deliverycurrently.

Much clinical work on RNA delivery has focused on RNAi or antisense, butthese systems can be adapted for delivery of RNA for implementing thepresent invention. References below to RNAi etc. should be readaccordingly.

Particle Delivery Systems and or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (μm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 μm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry(MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cassystem e.g., CRISPR enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845;5,855,913; 5,985,309; 5,543,158; and the publication by James E. Dahlmanand Carmen Barnes et al. Nature Nanotechnology (2014) published online11 May 2014, doi:10.1038/nnano.2014.84, concerning particles, methods ofmaking and using them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

Nanoparticles

CRISPR enzyme mRNA and guide RNA may be delivered simultaneously usingnanoparticles or lipid envelopes.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured nanoparticles with a poly(β-amino ester) (PBAE) coreenveloped by a phospholipid bilayer shell. These were developed for invivo mRNA delivery. The pH-responsive PBAE component was chosen topromote endosome disruption, while the lipid surface layer was selectedto minimize toxicity of the polycation core. Such are, therefore,preferred for delivering RNA of the present invention.

In one embodiment, nanoparticles based on self assembling bioadhesivepolymers are contemplated, which may be applied to oral delivery ofpeptides, intravenous delivery of peptides and nasal delivery ofpeptides, all to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Themolecular envelope technology involves an engineered polymer envelopewhich is protected and delivered to the site of the disease (see, e.g.,Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. MolPharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80;Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N. L., etal. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N. L., et al. J RamanSpect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9 andUchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses of about 5mg/kg are contemplated, with single or multiple doses, depending on thetarget tissue.

In one embodiment, nanoparticles that can deliver RNA to a cancer cellto stop tumor growth developed by Dan Anderson's lab at MIT may beused/and or adapted to the CRISPR Cas system of the present invention.In particular, the Anderson lab developed fully automated, combinatorialsystems for the synthesis, purification, characterization, andformulation of new biomaterials and nanoformulations. See, e.g., Alabiet al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6; Zhang etal., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., Nano Lett.2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012 Oct. 23;6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28; 6(8):6922-9 andLee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the CRISPR Cas system of the present invention. Inone aspect, the aminoalcohol lipidoid compounds are combined with anagent to be delivered to a cell or a subject to form microparticles,nanoparticles, liposomes, or micelles. The agent to be delivered by theparticles, liposomes, or micelles may be in the form of a gas, liquid,or solid, and the agent may be a polynucleotide, protein, peptide, orsmall molecule. The minoalcohol lipidoid compounds may be combined withother aminoalcohol lipidoid compounds, polymers (synthetic or natural),surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to formthe particles. These particles may then optionally be combined with apharmaceutical excipient to form a pharmaceutical composition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to the CRISPR Cassystem of the present invention.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe CRISPR Cas system of the present invention. Doses of about 0.01 toabout 1 mg per kg of body weight administered intravenously arecontemplated. Medications to reduce the risk of infusion-relatedreactions are contemplated, such as dexamethasone, acetampinophen,diphenhydramine or cetirizine, and ranitidine are contemplated. Multipledoses of about 0.3 mg per kilogram every 4 weeks for five doses are alsocontemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding CRISPR Cas to the liver. A dosage of about four doses of 6mg/kg of the LNP every two weeks may be contemplated. Tabernero et al.demonstrated that tumor regression was observed after the first 2 cyclesof LNPs dosed at 0.7 mg/kg, and by the end of 6 cycles the patient hadachieved a partial response with complete regression of the lymph nodemetastasis and substantial shrinkage of the liver tumors. A completeresponse was obtained after 40 doses in this patient, who has remainedin remission and completed treatment after receiving doses over 26months. Two patients with RCC and extrahepatic sites of diseaseincluding kidney, lung, and lymph nodes that were progressing followingprior therapy with VEGF pathway inhibitors had stable disease at allsites for approximately 8 to 12 months, and a patient with PNET andliver metastases continued on the extension study for 18 months (36doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12,pages 1286-2200, December 2011). Negatively charged polymers such as RNAmay be loaded into LNPs at low pH values (e.g., pH 4) where theionizable lipids display a positive charge. However, at physiological pHvalues, the LNPs exhibit a low surface charge compatible with longercirculation times. Four species of ionizable cationic lipids have beenfocused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA >DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR Cas encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificCRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA,DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMG orPEG-C-DOMG at 40:10:40:10 molar ratios). When required, 0.2% SP-DiOC18(Invitrogen, Burlington, Canada) may be incorporated to assess cellularuptake, intracellular delivery, and biodistribution. Encapsulation maybe performed by dissolving lipid mixtures comprised of cationiclipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanolto a final lipid concentration of 10 mmol/l. This ethanol solution oflipid may be added drop-wise to 50 mmol/l citrate, pH 4.0 to formmultilamellar vesicles to produce a final concentration of 30% ethanolvol/vol. Large unilamellar vesicles may be formed following extrusion ofmultilamellar vesicles through two stacked 80 nm Nuclepore polycarbonatefilters using the Extruder (Northern Lipids, Vancouver, Canada).Encapsulation may be achieved by adding RNA dissolved at 2 mg/ml in 50mmol/l citrate, pH 4.0 containing 30% ethanol vol/vol drop-wise toextruded preformed large unilamellar vesicles and incubation at 31° C.for 30 minutes with constant mixing to a final RNA/lipid weight ratio of0.06/1 wt/wt. Removal of ethanol and neutralization of formulationbuffer were performed by dialysis against phosphate-buffered saline(PBS), pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulosedialysis membranes. Nanoparticle size distribution may be determined bydynamic light scattering using a NICOMP 370 particle sizer, thevesicle/intensity modes, and Gaussian fitting (Nicomp Particle Sizing,Santa Barbara, Calif.). The particle size for all three LNP systems maybe ˜70 nm in diameter. RNA encapsulation efficiency may be determined byremoval of free RNA using VivaPureD MiniH columns (Sartorius StedimBiotech) from samples collected before and after dialysis. Theencapsulated RNA may be extracted from the eluted nanoparticles andquantified at 260 nm. RNA to lipid ratio was determined by measurementof cholesterol content in vesicles using the Cholesterol E enzymaticassay from Wako Chemicals USA (Richmond, Va.). In conjunction with theherein discussion of LNPs and PEG lipids, PEGylated liposomes or LNPsare likewise suitable for delivery of a CRISPR-Cas system or componentsthereof.

Preparation of large LNPs may be used/and or adapted from Rosin et al,Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011. Alipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano Z S, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at an RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other nanoparticles(particularly gold nanoparticles) are also contemplated as a means todelivery CRISPR-Cas system to intended targets. Significant data showthat AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs,based upon nucleic acid-functionalized gold nanoparticles, are useful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling nanoparticles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of CRISPR Cas is envisioned for deliveryin the self-assembling nanoparticles of Schiffelers et al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA nanoparticles maybe formed by using cyclodextrin-containing polycations. Typically,nanoparticles were formed in water at a charge ratio of 3 (+/−) and ansiRNA concentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted nanoparticles were modifiedwith Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5%(wt/vol) glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted nanoparticle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetednanoparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The nanoparticles consist of a synthetic deliverysystem containing: (1) a linear, cyclodextrin-based polymer (CDP), (2) ahuman transferrin protein (TF) targeting ligand displayed on theexterior of the nanoparticle to engage TF receptors (TFR) on the surfaceof the cancer cells, (3) a hydrophilic polymer (polyethylene glycol(PEG) used to promote nanoparticle stability in biological fluids), and(4) siRNA designed to reduce the expression of the RRM2 (sequence usedin the clinic was previously denoted siR2B+5). The TFR has long beenknown to be upregulated in malignant cells, and RRM2 is an establishedanti-cancer target. These nanoparticles (clinical version denoted asCALAA-01) have been shown to be well tolerated in multi-dosing studiesin non-human primates. Although a single patient with chronic myeloidleukaemia has been administered siRNAby liposomal delivery, Davis etal.'s clinical trial is the initial human trial to systemically deliversiRNA with a targeted delivery system and to treat patients with solidcancer. To ascertain whether the targeted delivery system can provideeffective delivery of functional siRNA to human tumours, Davis et al.investigated biopsies from three patients from three different dosingcohorts; patients A, B and C, all of whom had metastatic melanoma andreceived CALAA-01 doses of 18, 24 and 30 mg m-2 siRNA, respectively.Similar doses may also be contemplated for the CRISPR Cas system of thepresent invention. The delivery of the invention may be achieved withnanoparticles containing a linear, cyclodextrin-based polymer (CDP), ahuman transferrin protein (TF) targeting ligand displayed on theexterior of the nanoparticle to engage TF receptors (TFR) on the surfaceof the cancer cells and/or a hydrophilic polymer (for example,polyethylene glycol (PEG) used to promote nanoparticle stability inbiological fluids).

In terms of this invention, it is preferred to have one or morecomponents of CRISPR complex, e.g., CRISPR enzyme or mRNA or guide RNAor sgRNA or if present HDR template may be delivered using one or moreparticles or nanoparticles or lipid envelopes. Other delivery systems orvectors are may be used in conjunction with the nanoparticle aspects ofthe invention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, nanoparticles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm.

Nanoparticles encompassed in the present invention may be provided indifferent forms, e.g., as solid nanoparticles (e.g., metal such assilver, gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of nanoparticles, or combinations thereof. Metal,dielectric, and semiconductor nanoparticles may be prepared, as well ashybrid structures (e.g., core-shell nanoparticles). Nanoparticles madeof semiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft nanoparticles have been manufactured, and are withinthe scope of the present invention. A prototype nanoparticle ofsemi-solid nature is the liposome. Various types of liposomenanoparticles are currently used clinically as delivery systems foranticancer drugs and vaccines. Nanoparticles with one half hydrophilicand the other half hydrophobic are termed Janus particles and areparticularly effective for stabilizing emulsions. They can self-assembleat water/oil interfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingcomprising polymer conjugated to a surfactant, hydrophilic polymer orlipid. U.S. Pat. No. 6,007,845, incorporated herein by reference,provides particles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and contain abiologically active material. U.S. Pat. No. 5,855,913, incorporatedherein by reference, provides a particulate composition havingaerodynamically light particles having a tap density of less than 0.4g/cm3 with a mean diameter of between 5 μm and 30 μm, incorporating asurfactant on the surface thereof for drug delivery to the pulmonarysystem. U.S. Pat. No. 5,985,309, incorporated herein by reference,provides particles incorporating a surfactant and/or a hydrophilic orhydrophobic complex of a positively or negatively charged therapeutic ordiagnostic agent and a charged molecule of opposite charge for deliveryto the pulmonary system. U.S. Pat. No. 5,543,158, incorporated herein byreference, provides biodegradable injectable nanoparticles having abiodegradable solid core containing a biologically active material andpoly(alkylene glycol) moieties on the surface. WO2012135025 (alsopublished as US20120251560), incorporated herein by reference, describesconjugated polyethyleneimine (PEI) polymers and conjugatedaza-macrocycles (collectively referred to as “conjugated lipomer” or“lipomers”). In certain embodiments, it can be envisioned that suchmethods and materials of herein-cited documents, e.g., conjugatedlipomers, can be used in the context of the CRISPR-Cas system to achievein vitro, ex vivo and in vivo genomic perturbations to modify geneexpression, including modulation of protein expression.

In one embodiment, the nanoparticle may be epoxide-modifiedlipid-polymer, advantageously 7C1 (see, e.g., James E. Dahlman andCarmen Barnes et al. Nature Nanotechnology (2014) published online 11May 2014, doi:10.1038/nnano.2014.84). C71 was synthesized by reactingC15 epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14PEG2000 to produce nanoparticles (diameter between 35and 60 nm) that were stable in PBS solution for at least 40 days. Anepoxide-modified lipid-polymer may be utilized to deliver the CRISPR-Cassystem of the present invention to pulmonary, cardiovascular or renalcells, however, one of skill in the art may adapt the system to deliverto other target organs. Dosage ranging from about 0.05 to about 0.6mg/kg are envisioned. Dosages over several days or weeks are alsoenvisioned, with a total dosage of about 2 mg/kg.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by nanoparticle tracking analysis (NTA)and electron microscopy. Alvarez-Erviti et al. obtained 6-12 μg ofexosomes (measured based on protein concentration) per 106 cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 μF resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3 d after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNARVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or −] 15%, P<0.001 and61% [+ or −] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN-α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the CRISPR-Cas system of the present invention to therapeutictargets, especially neurodegenerative diseases. A dosage of about 100 to1000 mg of CRISPR Cas encapsulated in about 100 to 1000 mg of RVGexosomes may be contemplated for the present invention.

El-Andaloussi et al. (Nature Protocols 7, 2112-2126(2012)) discloses howexosomes derived from cultured cells can be harnessed for delivery ofRNA in vitro and in vivo. This protocol first describes the generationof targeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention.

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit(Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a finalconcentration of 2 mmol/ml. After adding HiPerFect transfection reagent,the mixture is incubated for 10 min at RT. In order to remove the excessof micelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of CRISPR Cas into exosomes may beconducted similarly to siRNA. The exosomes may be co-cultured withmonocytes and lymphocytes isolated from the peripheral blood of healthydonors. Therefore, it may be contemplated that exosomes containingCRISPR Cas may be introduced to monocytes and lymphocytes of andautologously reintroduced into a human. Accordingly, delivery oradministration according to the invention may be performed using plasmaexosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review). Liposomes can be madefrom several different types of lipids; however, phospholipids are mostcommonly used to generate liposomes as drug carriers. Although liposomeformation is spontaneous when a lipid film is mixed with an aqueoussolution, it can also be expedited by applying force in the form ofshaking by using a homogenizer, sonicator, or an extrusion apparatus(see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011,Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review). A liposome formulation may be mainly comprised of naturalphospholipids and lipids such as1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin,egg phosphatidylcholines and monosialoganglioside. Since thisformulation is made up of phospholipids only, liposomal formulationshave encountered many challenges, one of the ones being the instabilityin plasma. Several attempts to overcome these challenges have been made,specifically in the manipulation of the lipid membrane. One of theseattempts focused on the manipulation of cholesterol. Addition ofcholesterol to conventional formulations reduces rapid release of theencapsulated bioactive compound into the plasma or1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases thestability (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 forreview).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable. These particles allowdelivery of a transgene to the entire brain after an intravascularinjection. Without being bound by limitation, it is believed thatneutral lipid particles with specific antibodies conjugated to surfaceallow crossing of the blood brain barrier via endocytosis. Applicantpostulates utilizing Trojan Horse Liposomes to deliver the CRISPR familyof nucleases to the brain via an intravascular injection, which wouldallow whole brain transgenic animals without the need for embryonicmanipulation. About 1-5 g of DNA or RNA may be contemplated for in vivoadministration in liposomes.

In another embodiment, the CRISPR Cas system or components thereof maybe administered in liposomes, such as a stable nucleic-acid-lipidparticle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology,Vol. 23, No. 8, August 2005). Daily intravenous injections of about 1, 3or 5 mg/kg/day of a specific CRISPR Cas targeted in a SNALP arecontemplated. The daily treatment may be over about three days and thenweekly for about five weeks. In another embodiment, a specific CRISPRCas encapsulated SNALP) administered by intravenous injection to atdoses of about 1 or 2.5 mg/kg are also contemplated (see, e.g.,Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). The SNALPformulation may contain the lipids 3-N-[(wmethoxypoly(ethylene glycol)2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size. In yet another embodiment, a SNALP may comprisesynthetic cholesterol (Sigma-Aldrich, St Louis, Mo., USA),dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala.,USA), 3-N-[(w-methoxy poly(ethyleneglycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g., Geisbert et al.,Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kg total CRISPR Casper dose administered as, for example, a bolus intravenous infusion maybe contemplated. In yet another embodiment, a SNALP may comprisesynthetic cholesterol (Sigma-Aldrich),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar LipidsInc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N:N-dimethyl)aminopropane(DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)).Formulations used for in vivo studies may comprise a final lipid/RNAmass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC)—both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR 01 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≥0.4 mg/kg;all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-Ira were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Niotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mMKH2PO4, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 μmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the CRISPR Cas system of the presentinvention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate CRISPR Cas system or components thereof ornucleic acid molecule(s) coding therefor e.g., similar to SiRNA (see,e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533), and hencemay be employed in the practice of the invention. A preformed vesiclewith the following lipid composition may be contemplated: amino lipid,distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11+0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the CRISPR Cas RNA. Particles containing the highlypotent amino lipid 16 may be used, in which the molar ratio of the fourlipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5)which may be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume:29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the CRISPR Cassystem of the present invention to form lipid nanoparticles (LNPs).Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 andcolipids disteroylphosphatidyl choline, cholesterol, and PEG-DMG may beformulated with CRISPR Cas instead of siRNA (see, e.g., Novobrantseva,Molecular Therapy-Nucleic Acids (2012) 1, e4; doi:10.1038/mtna.2011.3)using a spontaneous vesicle formation procedure. The component molarratio may be about 50/10/38.5/1.5 (DLin-KC 2-DMA orC12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG). The finallipid:siRNA weight ratio may be ˜12:1 and 9:1 in the case ofDLin-KC2-DMA and C12-200 lipid nanoparticles (LNPs), respectively. Theformulations may have mean particle diameters of ˜80 nm with >90%entrapment efficiency. A 3 mg/kg dose may be contemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The CRISPR Cas system or components thereof or nucleic acid molecule(s)coding therefor may be delivered encapsulated in PLGA Microspheres suchas that further described in US published applications 20130252281 and20130245107 and 20130244279 (assigned to Moderna Therapeutics) whichrelate to aspects of formulation of compositions comprising modifiednucleic acid molecules which may encode a protein, a protein precursor,or a partially or fully processed form of the protein or a proteinprecursor. The formulation may have a molar ratio 50:10:38.5:1.5-3.0(cationic lipid:fusogenic lipid:cholesterol:PEG lipid). The PEG lipidmay be selected from, but is not limited to PEG-c-DOMG, PEG-DMG. Thefusogenic lipid may be DSPC. See also, Schrum et al., Delivery andFormulation of Engineered Nucleic Acids, US published application20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesised from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN—P(O2)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied.

US Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropylenimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumouractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of CRISPR Cas system(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of CRISPR Cas system(s) or component(s)thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116). However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines.

-   -   (1) One day before treatment, plate 1×105 cells per well in a        48-well plate.    -   (2) On the day of treatment, dilute purified +36 GFP protein in        serumfree media to a final concentration 200 nM. Add RNA to a        final concentration of 50 nM. Vortex to mix and incubate at room        temperature for 10 min.    -   (3) During incubation, aspirate media from cells and wash once        with PBS.    -   (4) Following incubation of +36 GFP and RNA, add the protein-RNA        complexes to cells.    -   (5) Incubate cells with complexes at 37° C. for 4 h.    -   (6) Following incubation, aspirate the media and wash three        times with 20 U/mL heparin PBS. Incubate cells with        serum-containing media for a further 48 h or longer depending        upon the assay for activity.    -   (7) Analyze cells by immunoblot, qPCR, phenotypic assay, or        other appropriate method.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications.

-   -   (1) One day before treatment, plate 1×105 per well in a 48-well        plate.    -   (2) On the day of treatment, dilute purified        36 GFP protein in serumfree media to a final concentration 2 mM.        Add 1 mg of plasmid DNA. Vortex to mix and incubate at room        temperature for 10 min.    -   (3) During incubation, aspirate media from cells and wash once        with PBS.    -   (4) Following incubation of        36 GFP and plasmid DNA, gently add the protein-DNA complexes to        cells.    -   (5) Incubate cells with complexes at 37 C for 4 h.    -   (6) Following incubation, aspirate the media and wash with PBS.        Incubate cells in serum-containing media and incubate for a        further 24-48 h.    -   (7) Analyze plasmid delivery (e.g., by plasmid-driven gene        expression) as appropriate.

See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106,6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe CRISPR Cas system of the present invention. These systems of Dr. Luiand documents herein in inconjunction with herein teachints can beemployed in the delivery of CRISPR Cas system(s) or component(s) thereofor nucleic acid molecule(s) coding therefor.

Cell Penetrating Peptides ((PPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins,nanoparticles, liposomes, chromophores, small molecules and radioactivematerials. In aspects of the invention, the cargo may also comprise anycomponent of the CRISPR Cas system or the entire functional CRISPR Cassystem. Aspects of the present invention further provide methods fordelivering a desired cargo into a subject comprising: (a) preparing acomplex comprising the cell penetrating peptide of the present inventionand a desired cargo, and (b) orally, intraarticularly,intraperitoneally, intrathecally, intrarterially, intranasally,intraparenchymally, subcutaneously, intramuscularly, intravenously,dermally, intrarectally, or topically administering the complex to asubject. The cargo is associated with the peptides either throughchemical linkage via covalent bonds or through non-covalentinteractions.

The function of the CPPs are to deliver the cargo into cells, a processthat commonly occurs through endocytosis with the cargo delivered to theendosomes of living mammalian cells. Cell-penetrating peptides are ofdifferent sizes, amino acid sequences, and charges but all CPPs have onedistinct characteristic, which is the ability to translocate the plasmamembrane and facilitate the delivery of various molecular cargoes to thecytoplasm or an organelle. CPP translocation may be classified intothree main entry mechanisms: direct penetration in the membrane,endocytosis-mediated entry, and translocation through the formation of atransitory structure. CPPs have found numerous applications in medicineas drug delivery agents in the treatment of different diseases includingcancer and virus inhibitors, as well as contrast agents for celllabeling. Examples of the latter include acting as a carrier for GFP,MRI contrast agents, or quantum dots. CPPs hold great potential as invitro and in vivo delivery vectors for use in research and medicine.CPPs typically have an amino acid composition that either contains ahigh relative abundance of positively charged amino acids such as lysineor arginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. Thesetwo types of structures are referred to as polycationic or amphipathic,respectively. A third class of CPPs are the hydrophobic peptides,containing only apolar residues, with low net charge or have hydrophobicamino acid groups that are crucial for cellular uptake. One of theinitial CPPs discovered was the trans-activating transcriptionalactivator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) which wasfound to be efficiently taken up from the surrounding media by numerouscell types in culture. Since then, the number of known CPPs has expandedconsiderably and small molecule synthetic analogues with more effectiveprotein transduction properties have been generated. CPPs include butare not limited to Penetratin, Tat (48-60), Transportan, and (R-AhX-R4)(Ahx=aminohexanoyl) (SEQ ID NO: 194).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8,614,194 and 8,044,019. CPPscan be used to deliver the CRISPR-Cas system or components thereof. ThatCPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged nanoparticles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the CRISPR Cas system or component(s) thereof or nucleicacid molecule(s) coding therefor. For example, US Patent Publication20110195123 discloses an implantable medical device which elutes a druglocally and in prolonged period is provided, including several types ofsuch a device, the treatment modes of implementation and methods ofimplantation. The device comprising of polymeric substrate, such as amatrix for example, that is used as the device body, and drugs, and insome cases additional scaffolding materials, such as metals oradditional polymers, and materials to enhance visibility and imaging. Animplantable delivery device can be advantageous in providing releaselocally and over a prolonged period, where drug is released directly tothe extracellular matrix (ECM) of the diseased area such as tumor,inflammation, degeneration or for symptomatic objectives, or to injuredsmooth muscle cells, or for prevention. One kind of drug is RNA, asdisclosed above, and this system may be used/and or adapted to theCRISPR Cas system of the present invention. The modes of implantation insome embodiments are existing implantation procedures that are developedand used today for other treatments, including brachytherapy and needlebiopsy. In such cases the dimensions of the new implant described inthis invention are similar to the original implant. Typically a fewdevices are implanted during the same treatment procedure.

US Patent Publication 20110195123 provides a drug delivery implantableor insertable system, including systems applicable to a cavity such asthe abdominal cavity and/or any other type of administration in whichthe drug delivery system is not anchored or attached, comprising abiostable and/or degradable and/or bioabsorbable polymeric substrate,which may for example optionally be a matrix. It should be noted thatthe term “insertion” also includes implantation. The drug deliverysystem is preferably implemented as a “Loder” as described in US PatentPublication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m³ to 1000 mm³, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system of US Patent Publication 20110195123 isoptionally associated with sensing and/or activation appliances that areoperated at and/or after implantation of the device, by non and/orminimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of US Patent Publication 20110195123, thesite for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group consisting of(as non-limiting examples only, as optionally any site within the bodymay be suitable for implanting a Loder): 1. brain at degenerative siteslike in Parkinson or Alzheimer disease at the basal ganglia, white andgray matter; 2. spine as in the case of amyotrophic lateral sclerosis(ALS); 3. uterine cervix to prevent HPV infection; 4. active and chronicinflammatory joints; 5. dermis as in the case of psoriasis; 6.sympathetic and sensoric nervous sites for analgesic effect; 7. Intraosseous implantation; 8. acute and chronic infection sites; 9. Intravaginal; 10. Inner ear-auditory system, labyrinth of the inner ear,vestibular system; 11. Intra tracheal; 12. Intra-cardiac; coronary,epicardiac; 13. urinary bladder; 14. biliary system; 15. parenchymaltissue including and not limited to the kidney, liver, spleen; 16. lymphnodes; 17. salivary glands; 18. dental gums; 19. Intra-articular (intojoints); 20. Intra-ocular; 21. Brain tissue; 22. Brain ventricles; 23.Cavities, including abdominal cavity (for example but withoutlimitation, for ovary cancer); 24. Intra esophageal and 25. Intrarectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of US Patent Publication 20110195123, thedrug preferably comprises a RNA, for example for localized cancer casesin breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the CRISPR Cas system of the presentinvention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the CRISPR Cas system of the present invention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the CRISPR Cas system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used/and or adapted to the CRISPR Cas system of the presentinvention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Implantable device technology herein discussed can be employed withherein teachings and hence by this disclosure and the knowledge in theart, CRISPR-Cas system or components thereof or nucleic acid moleculesthereof or encoding or providing components may be delivered via animplantable device.

Fluid Delivery Device Methods

In another embodiment, a fluid delivery device with an array of needles(see, e.g., US Patent Publication No. 20110230839 assigned to the FredHutchinson Cancer Research Center) may be contemplated for delivery ofCRISPR Cas to solid tissue. A device of US Patent Publication No.20110230839 for delivery of a fluid to a solid tissue may comprise aplurality of needles arranged in an array; a plurality of reservoirs,each in fluid communication with a respective one of the plurality ofneedles; and a plurality of actuators operatively coupled to respectiveones of the plurality of reservoirs and configured to control a fluidpressure within the reservoir. In certain embodiments each of theplurality of actuators may comprise one of a plurality of plungers, afirst end of each of the plurality of plungers being received in arespective one of the plurality of reservoirs, and in certain furtherembodiments the plungers of the plurality of plungers are operativelycoupled together at respective second ends so as to be simultaneouslydepressable. Certain still further embodiments may comprise a plungerdriver configured to depress all of the plurality of plungers at aselectively variable rate. In other embodiments each of the plurality ofactuators may comprise one of a plurality of fluid transmission lineshaving first and second ends, a first end of each of the plurality offluid transmission lines being coupled to a respective one of theplurality of reservoirs. In other embodiments the device may comprise afluid pressure source, and each of the plurality of actuators comprisesa fluid coupling between the fluid pressure source and a respective oneof the plurality of reservoirs. In further embodiments the fluidpressure source may comprise at least one of a compressor, a vacuumaccumulator, a peristaltic pump, a master cylinder, a microfluidic pump,and a valve. In another embodiment, each of the plurality of needles maycomprise a plurality of ports distributed along its length.

Patient-Specific Screening Methods

A CRISPR-Cas system that targets nucleotide, e.g., trinucleotide repeatscan be used to screen patients or patent samples for the presence ofsuch repeats. The repeats can be the target of the RNA of the CRISPR-Cassystem, and if there is binding thereto by the CRISPR-Cas system, thatbinding can be detected, to thereby indicate that such a repeat ispresent. Thus, a CRISPR-Cas system can be used to screen patients orpatient samples for the presence of the repeat. The patient can then beadministered suitable compound(s) to address the condition; or, can beadministered a CRISPR-Cas system to bind to and cause insertion,deletion or mutation and alleviate the condition.

Bone

Oakes and Lieberman (Clin Orthop Relat Res. 2000 October; (379Suppl):S101-12) discusses delivery of genes to the bone. By transferringgenes into cells at a specific anatomic site, the osteoinductiveproperties of growth factors can be used at physiologic doses for asustained period to facilitate a more significant healing response. Thespecific anatomic site, the quality of the bone, and the soft-tissueenvelope, influences the selection of the target cells for regional genetherapy. Gene therapy vectors delivered to a treatment site inosteoconductive carriers have yielded promising results. Severalinvestigators have shown exciting results using ex vivo and in vivoregional gene therapy in animal models. Such a system may be used/and oradapted to the CRISPR Cas system for delivery to the bone.

Targeted Deletion, Therapeutic Applications

Targeted deletion of genes is preferred. Preferred are, therefore, genesinvolved in cholesterol biosynthesis, fatty acid biosynthesis, and othermetabolic disorders, genes encoding mis-folded proteins involved inamyloid and other diseases, oncogenes leading to cellulartransformation, latent viral genes, and genes leading todominant-negative disorders, amongst other disorders. As exemplifiedhere, Applicants prefer gene delivery of a CRISPR-Cas system to the eye(ocular), ear (auditory), epithelial, hematopoetic, or another tissue ofa subject or a patient in need thereof, suffering from metabolicdisorders, amyloidosis and protein-aggregation related diseases,cellular transformation arising from genetic mutations andtranslocations, dominant negative effects of gene mutations, latentviral infections, and other related symptoms, using either viral ornanoparticle delivery system. Therapeutic applications of the CRISPR-Cassystem include hereditary ocular diseases, including but not limited toretinitis pigmentosa, achromatopsia, macular degeneration, glaucoma,etc. A list of ocular diseases is provided herein (see section entitledocular gene therapy).

As an example, chronic infection by HIV-1 may be treated or prevented.In order to accomplish this, one may generate CRISPR-Cas guide RNAs thattarget the vast majority of the HIV-1 genome while taking into accountHIV-1 strain variants for maximal coverage and effectiveness. One mayaccomplish delivery of the CRISPR-Cas system by conventional adenoviralor lentiviral-mediated infection of the host immune system. Depending onapproach, host immune cells could be a) isolated, transduced withCRISPR-Cas, selected, and re-introduced in to the host or b) transducedin vivo by systemic delivery of the CRISPR-Cas system. The firstapproach allows for generation of a resistant immune population whereasthe second is more likely to target latent viral reservoirs within thehost. This is discussed in more detail in the Examples section.

In another example, US Patent Publication No. 20130171732 assigned toSangamo BioSciences, Inc. relates to insertion of an anti-HIV transgeneinto the genome, methods of which may be applied to the CRISPR Cassystem of the present invention. In another embodiment, the CXCR4 genemay be targeted and the TALE system of US Patent Publication No.20100291048 assigned to Sangamo BioSciences, Inc. may be modified to theCRISPR Cas system of the present invention. The method of US PatentPublication Nos. 20130137104 and 20130122591 assigned to SangamoBioSciences, Inc. and US Patent Publication No. 20100146651 assigned toCellectis may be more generally applicable for transgene expression asit involves modifying a hypoxanthine-guanine phosphoribosyltransferase(HPRT) locus for increasing the frequency of gene modification.

It is also envisaged that the present invention generates a geneknockout cell library. Each cell may have a single gene knocked out.

One may make a library of ES cells where each cell has a single geneknocked out, and the entire library of ES cells will have every singlegene knocked out. This library is useful for the screening of genefunction in cellular processes as well as diseases. To make this celllibrary, one may integrate Cas9 driven by an inducible promoter (e.g.doxycycline inducible promoter) into the ES cell. In addition, one mayintegrate a single guide RNA targeting a specific gene in the ES cell.To make the ES cell library, one may simply mix ES cells with a libraryof genes encoding guide RNAs targeting each gene in the human genome.One may first introduce a single BxB1 attB site into the AAVS1 locus ofthe human ES cell. Then one may use the BxB1 integrase to facilitate theintegration of individual guide RNA genes into the BxB1 attB site inAAVS1 locus. To facilitate integration, each guide RNA gene may becontained on a plasmid that carries of a single attP site. This way BxB1will recombine the attB site in the genome with the attP site on theguide RNA containing plasmid. To generate the cell library, one may takethe library of cells that have single guide RNAs integrated and induceCas9 expression. After induction, Cas9 mediates double strand break atsites specified by the guide RNA.

Chronic administration of protein therapeutics may elicit unacceptableimmune responses to the specific protein. The immunogenicity of proteindrugs can be ascribed to a few immunodominant helper T lymphocyte (HTL)epitopes. Reducing the MHC binding affinity of these HTL epitopescontained within these proteins can generate drugs with lowerimmunogenicity (Tangri S, et al. (“Rationally engineered therapeuticproteins with reduced immunogenicity” J Immunol. 2005 Mar. 15;174(6):3187-96.) In the present invention, the immunogenicity of theCRISPR enzyme in particular may be reduced following the approach firstset out in Tangri et al with respect to erythropoietin and subsequentlydeveloped. Accordingly, directed evolution or rational design may beused to reduce the immunogenicity of the CRISPR enzyme (for instance aCas9) in the host species (human or other species).

Applicants have shown targeted in vivo cleavage using in an exemplaryembodiment 3 guideRNAs of interest and are able to visualize efficientDNA cleavage in vivo occurring only in a small subset of cells. (seee.g., Example 1) In particular, this illustrates that specific targetingin higher organisms such as mammals can also be achieved. It alsohighlights multiplex aspect in that multiple guide sequences (i.e.separate targets) can be used simultaneously (in the sense ofco-delivery). In other words, Applicants used a multiple approach, withseveral different sequences targeted at the same time, butindependently.

Blood

The present invention also contemplates delivering the CRISPR-Cas systemto the blood.

The plasma exosomes of Wahlgren et al. (Nucleic Acids Research, 2012,Vol. 40, No. 17 e130) may be utilized to deliver the CRISPR Cas systemto the blood. Other means of delivery or RNA are also preferred, such asvia nanoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F.,Mei, Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-likenanoparticles for small interfering RNA

The CRISPR Cas system of the present invention is also contemplated totreat hemoglobinopathies, such as thalassemias and sickle cell disease.See, e.g., International Patent Publication No. WO 2013/126794 forpotential targets that may be targeted by the CRISPR Cas system of thepresent invention. Drakopoulou, “Review Article, The Ongoing Challengeof Hematopoietic Stem Cell-Based Gene Therapy for β-Thalassemia,” StemCells International, Volume 2011, Article ID 987980, 10 pages,doi:10.4061/2011/987980, incorporated herein by reference along with thedocuments it cites, as if set out in full, discuss modifying HSCs usinga lentivirus that delivers a gene for β-globin or γ-globin. In contrastto using lentivirus, with the knowledge in the art and the teachings inthis disclosure, the skilled person can correct HSCs as to β-Thalassemiausing a CRISPR-Cas9 system that targets and corrects the mutation (e.g.,with a suitable HDR template that delivers a coding sequence forβ-globin or γ-globin, advantageously non-sickling β-globin or γ-globin);specifically, the sgRNA can target mutation that give rise toβ-Thalassemia, and the HDR can provide coding for proper expression ofβ-globin or γ-globin. In this regard mention is made of: Cavazzana,“Outcomes of Gene Therapy for β-Thalassemia Major via Transplantation ofAutologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviralβ^(A-T87Q)-Globin Vector.” tif2014.org/abstractFiles/Jean %20Antoine%20Ribeil_Abstract.pdf; Cavazzana-Calvo, “Transfusion independence andHMGA2 activation after gene therapy of human β-thalassaemia”, Nature467, 318-322 (16 Sep. 2010) doi:10.1038/nature09328; Nienhuis,“Development of Gene Therapy for Thalassemia, Cold Spring HarborPerpsectives in Medicine, doi: 10.1101/cshperspect.a011833 (2012),LentiGlobin BB305, a lentiviral vector containing an engineered β-globingene (βA-T87Q); and Xie et al., “Seamless gene correction ofβ-thalassaemia mutations in patient-specific iPSCs using CRISPR/Cas9 andpiggyback” Genome Research gr.173427.114 (2014) 24: 1526-1533 (ColdSpring Harbor Laboratory Press); that is the subject of Cavazzana workinvolving human β-thalassaemia and the subject of the Xie work, are allincorporated herein by reference, together with all documents citedtherein or associated therewith. In the instant invention, the HDRtemplate can provide for the HSC to express an engineered β-globin gene(e.g., βA-T87Q), or β-globin as in Xie. Sickle cell anemia is anautosomal recessive genetic disease in which red blood cells becomesickle-shaped. It is caused by a single base substitution in theβ-globin gene, which is located on the short arm of chromosome 11. As aresult, valine is produced instead of glutamic acid causing theproduction of sickle hemoglobin (HbS). This results in the formation ofa distorted shape of the erythrocytes. Due to this abnormal shape, smallblood vessels can be blocked, causing serious damage to the bone, spleenand skin tissues. This may lead to episodes of pain, frequentinfections, hand-foot syndrome or even multiple organ failure. Thedistorted erythrocytes are also more susceptible to hemolysis, whichleads to serious anemia. As in the case of β-thalassaemia, sickle cellanemia can be corrected by modifying HSCs with the CRISPR/Cas9 system.The system allows the specific editing of the cell's genome by cuttingits DNA and then letting it repair itself. The Cas9 protein is insertedand directed by a RNA guide to the mutated point and then it cuts theDNA at that point. Simultaneously, a healthy version of the sequence isinserted. This sequence is used by the cell's own repair system to fixthe induced cut. In this way, the CRISPR/Cas9 allows the correction ofthe mutation in the previously obtained stem cells. With the knowledgein the art and the teachings in this disclosure, the skilled person cancorrect HSCs as to sickle cell anemia using a CRISPR-Cas9 system thattargets and corrects the mutation (e.g., with a suitable HDR templatethat delivers a coding sequence for β-globin, advantageouslynon-sickling β-globin); specifically, the sgRNA can target mutation thatgive rise to sickle cell anemia, and the HDR can provide coding forproper expression of β-globin.

US Patent Publication Nos. 20110225664, 20110091441, 20100229252,20090271881 and 20090222937 assigned to Cellectis, relates to CREIvariants, wherein at least one of the two I-CreI monomers has at leasttwo substitutions, one in each of the two functional subdomains of theLAGLIDADG core domain (SEQ ID NO: 41) situated respectively frompositions 26 to 40 and 44 to 77 of I-CreI, said variant being able tocleave a DNA target sequence from the human interleukin-2 receptor gammachain (IL2RG) gene also named common cytokine receptor gamma chain geneor gamma C gene. The target sequences identified in US PatentPublication Nos. 20110225664, 20110091441, 20100229252, 20090271881 and20090222937 may be utilized for the CRISPR Cas system of the presentinvention.

Severe Combined Immune Deficiency (SCID) results from a defect inlymphocytes T maturation, always associated with a functional defect inlymphocytes B (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56,585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109). Overallincidence is estimated to 1 in 75 000 births. Patients with untreatedSCID are subject to multiple opportunist micro-organism infections, anddo generally not live beyond one year. SCID can be treated by allogenichematopoietic stem cell transfer, from a familial donor.Histocompatibility with the donor can vary widely. In the case ofAdenosine Deaminase (ADA) deficiency, one of the SCID forms, patientscan be treated by injection of recombinant Adenosine Deaminase enzyme.

Since the ADA gene has been shown to be mutated in SCID patients(Giblett et al., Lancet, 1972, 2, 1067-1069), several other genesinvolved in SCID have been identified (Cavazzana-Calvo et al., Annu.Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005, 203,98-109). There are four major causes for SCID: (i) the most frequentform of SCID, SCID-X1 (X-linked SCID or X-SCID), is caused by mutationin the IL2RG gene, resulting in the absence of mature T lymphocytes andNK cells. IL2RG encodes the gamma C protein (Noguchi, et al., Cell,1993, 73, 147-157), a common component of at least five interleukinreceptor complexes. These receptors activate several targets through theJAK3 kinase (Macchi et al., Nature, 1995, 377, 65-68), whichinactivation results in the same syndrome as gamma C inactivation; (ii)mutation in the ADA gene results in a defect in purine metabolism thatis lethal for lymphocyte precursors, which in turn results in the quasiabsence of B, T and NK cells; (iii) V(D)J recombination is an essentialstep in the maturation of immunoglobulins and T lymphocytes receptors(TCRs). Mutations in Recombination Activating Gene 1 and 2 (RAG1 andRAG2) and Artemis, three genes involved in this process, result in theabsence of mature T and B lymphocytes; and (iv) Mutations in other genessuch as CD45, involved in T cell specific signaling have also beenreported, although they represent a minority of cases (Cavazzana-Calvoet al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol.Rev., 2005, 203, 98-109).

Since when their genetic bases have been identified, the different SCIDforms have become a paradigm for gene therapy approaches (Fischer etal., Immunol. Rev., 2005, 203, 98-109) for two major reasons. An ex vivotreatment is envisioned. Hematopoietic Stem Cells (HSCs) can berecovered from bone marrow, and keep their pluripotent properties for afew cell divisions. Therefore, they can be treated in vitro, and thenreinjected into the patient, where they repopulate the bone marrow.Since the maturation of lymphocytes is impaired in SCID patients,corrected cells have a selective advantage. Therefore, a small number ofcorrected cells can restore a functional immune system. This hypothesishas been validated in other systems by (i) the partial restoration ofimmune functions associated with the reversion of mutations in SCIDpatients (Hirschhorn et al., Nat. Genet., 1996, 13, 290-295; Stephan etal., N. Engl. J. Med., 1996, 335, 1563-1567; Bousso et al., Proc. Natl.,Acad. Sci. USA, 2000, 97, 274-278; Wada et al., Proc. Natl. Acad. Sci.USA, 2001, 98, 8697-8702; Nishikomori et al., Blood, 2004, 103,4565-4572), (ii) the correction of SCID-X1 deficiencies in vitro inhematopoietic cells (Candotti et al., Blood, 1996, 87, 3097-3102;Cavazzana-Calvo et al., Blood, 1996, Blood, 88, 3901-3909; Taylor etal., Blood, 1996, 87, 3103-3107; Hacein-Bey et al., Blood, 1998, 92,4090-4097), (iii) the correction of SCID-X1 (Soudais et al., Blood,2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), JAK-3(Bunting et al., Nat. Med., 1998, 4, 58-64; Bunting et al., Hum. GeneTher., 2000, 11, 2353-2364) and RAG2 (Yates et al., Blood, 2002, 100,3942-3949) deficiencies in vivo in animal models and (iv) by the resultof gene therapy clinical trials (Cavazzana-Calvo et al., Science, 2000,288, 669-672; Aiuti et al., Nat. Med., 2002; 8, 423-425; Gaspar et al.,Lancet, 2004, 364, 2181-2187). From this disclosure, one can use aCRISPR-Cas9 system that targets and one or more of the mutationsassociated with SCID, for instance a CRISPR-Cas9 system having sgRNA(s)and HDR template(s) that respectively targets mutation of IL2RG thatgive rise to SCID and provide corrective expression of the gamma Cprotein.

US Patent Publication No. 20110182867 assigned to the Children's MedicalCenter Corporation and the President and Fellows of Harvard Collegerelates to methods and uses of modulating fetal hemoglobin expression(HbF) in a hematopoietic progenitor cells via inhibitors of BCL11Aexpression or activity, such as RNAi and antibodies. The targetsdisclosed in US Patent Publication No. 20110182867, such as BCL11A, maybe targeted by the CRISPR Cas system of the present invention formodulating fetal hemoglobin expression. See also Bauer et al. (Science11 Oct. 2013: Vol. 342 no. 6155 pp. 253-257) and Xu et al. (Science 18Nov. 2011: Vol. 334 no. 6058 pp. 993-996) for additional BCL11A targets.

Ears

The present invention also contemplates delivering the CRISPR-Cas systemto one or both ears.

Researchers are looking into whether gene therapy could be used to aidcurrent deafness treatments—namely, cochlear implants. Deafness is oftencaused by lost or damaged hair cells that cannot relay signals toauditory neurons. In such cases, cochlear implants may be used torespond to sound and transmit electrical signals to the nerve cells. Butthese neurons often degenerate and retract from the cochlea as fewergrowth factors are released by impaired hair cells.

US patent application 20120328580 describes injection of apharmaceutical composition into the ear (e.g., auricularadministration), such as into the luminae of the cochlea (e.g., theScala media, Sc vestibulae, and Sc tympani), e.g., using a syringe,e.g., a single-dose syringe. For example, one or more of the compoundsdescribed herein can be administered by intratympanic injection (e.g.,into the middle ear), and/or injections into the outer, middle, and/orinner ear. Such methods are routinely used in the art, for example, forthe administration of steroids and antibiotics into human ears.Injection can be, for example, through the round window of the ear orthrough the cochlear capsule. Other inner ear administration methods areknown in the art (see, e.g., Salt and Plontke, Drug Discovery Today,10:1299-1306, 2005).

In another mode of administration, the pharmaceutical composition can beadministered in situ, via a catheter or pump. A catheter or pump can,for example, direct a pharmaceutical composition into the cochlearluminae or the round window of the ear and/or the lumen of the colon.Exemplary drug delivery apparatus and methods suitable for administeringone or more of the compounds described herein into an ear, e.g., a humanear, are described by McKenna et al., (U.S. Publication No.2006/0030837) and Jacobsen et al., (U.S. Pat. No. 7,206,639). In someembodiments, a catheter or pump can be positioned, e.g., in the ear(e.g., the outer, middle, and/or inner ear) of a patient during asurgical procedure. In some embodiments, a catheter or pump can bepositioned, e.g., in the ear (e.g., the outer, middle, and/or inner ear)of a patient without the need for a surgical procedure.

Alternatively or in addition, one or more of the compounds describedherein can be administered in combination with a mechanical device suchas a cochlear implant or a hearing aid, which is worn in the outer ear.An exemplary cochlear implant that is suitable for use with the presentinvention is described by Edge et al., (U.S. Publication No.2007/0093878).

In some embodiments, the modes of administration described above may becombined in any order and can be simultaneous or interspersed.

Alternatively or in addition, the present invention may be administeredaccording to any of the Food and Drug Administration approved methods,for example, as described in CDER Data Standards Manual, version number004 (which is available at fda.give/cder/dsm/DRG/drg00301.htm).

In general, the cell therapy methods described in US patent application20120328580 can be used to promote complete or partial differentiationof a cell to or towards a mature cell type of the inner ear (e.g., ahair cell) in vitro. Cells resulting from such methods can then betransplanted or implanted into a patient in need of such treatment. Thecell culture methods required to practice these methods, includingmethods for identifying and selecting suitable cell types, methods forpromoting complete or partial differentiation of selected cells, methodsfor identifying complete or partially differentiated cell types, andmethods for implanting complete or partially differentiated cells aredescribed below.

Cells suitable for use in the present invention include, but are notlimited to, cells that are capable of differentiating completely orpartially into a mature cell of the inner ear, e.g., a hair cell (e.g.,an inner and/or outer hair cell), when contacted, e.g., in vitro, withone or more of the compounds described herein. Exemplary cells that arecapable of differentiating into a hair cell include, but are not limitedto stem cells (e.g., inner ear stem cells, adult stem cells, bone marrowderived stem cells, embryonic stem cells, mesenchymal stem cells, skinstem cells, iPS cells, and fat derived stem cells), progenitor cells(e.g., inner ear progenitor cells), support cells (e.g., Deiters' cells,pillar cells, inner phalangeal cells, tectal cells and Hensen's cells),and/or germ cells. The use of stem cells for the replacement of innerear sensory cells is described in Li et al., (U.S. Publication No.2005/0287127) and Li et al., (U.S. patent Ser. No. 11/953,797). The useof bone marrow derived stem cells for the replacement of inner earsensory cells is described in Edge et al., PCT/US2007/084654. iPS cellsare described, e.g., at Takahashi et al., Cell, Volume 131, Issue 5,Pages 861-872 (2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006);Okita et al., Nature 448, 260-262 (2007); Yu, J. et al., Science318(5858):1917-1920 (2007); Nakagawa et al., Nat. Biotechnol. 26:101-106(2008); and Zaehres and Scholer, Cell 131(5):834-835 (2007).

Such suitable cells can be identified by analyzing (e.g., qualitativelyor quantitatively) the presence of one or more tissue specific genes.For example, gene expression can be detected by detecting the proteinproduct of one or more tissue-specific genes. Protein detectiontechniques involve staining proteins (e.g., using cell extracts or wholecells) using antibodies against the appropriate antigen. In this case,the appropriate antigen is the protein product of the tissue-specificgene expression. Although, in principle, a first antibody (i.e., theantibody that binds the antigen) can be labeled, it is more common (andimproves the visualization) to use a second antibody directed againstthe first (e.g., an anti-IgG). This second antibody is conjugated eitherwith fluorochromes, or appropriate enzymes for colorimetric reactions,or gold beads (for electron microscopy), or with the biotin-avidinsystem, so that the location of the primary antibody, and thus theantigen, can be recognized.

The CRISPR Cas molecules of the present invention may be delivered tothe ear by direct application of pharmaceutical composition to the outerear, with compositions modified from US Published application,20110142917. In some embodiments the pharmaceutical composition isapplied to the ear canal. Delivery to the ear may also be referred to asaural or otic delivery.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference.

Delivery systems aimed specifically at the enhanced and improveddelivery of siRNA into mammalian cells have been developed, (see, forexample, Shen et al FEBS Let. 2003, 539:111-114; Xia et al., Nat.Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision. 2003, 9:210-216; Sorensen et al., J. Mol. Biol. 2003, 327: 761-766; Lewis etal., Nat. Gen. 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31, 11:2717-2724) and may be applied to the present invention. siRNA hasrecently been successfully used for inhibition of gene expression inprimates (see for example. Tolentino et al., Retina 24(4):660 which mayalso be applied to the present invention.

Qi et al. discloses methods for efficient siRNA transfection to theinner ear through the intact round window by a novel proteidic deliverytechnology which may be applied to the CRISPR Cas system of the presentinvention (see, e.g., Qi et al., Gene Therapy (2013), 1-9). Inparticular, a TAT double stranded RNA-binding domains (TAT-DRBDs), whichcan transfect Cy3-labeled siRNA into cells of the inner ear, includingthe inner and outer hair cells, Crista ampullaris, macula utriculi andmacula sacculi, through intact round-window permeation was successfulfor delivering double stranded siRNAs in vivo for treating various innerear ailments and preservation of hearing function. About 40 μl of 10 mMRNA may be contemplated as the dosage for administration to the ear.

According to Rejali et al. (Hear Res. 2007 June; 228(1-2):180-7),cochlear implant function can be improved by good preservation of thespiral ganglion neurons, which are the target of electrical stimulationby the implant and brain derived neurotrophic factor (BDNF) haspreviously been shown to enhance spiral ganglion survival inexperimentally deafened ears. Rejali et al. tested a modified design ofthe cochlear implant electrode that includes a coating of fibroblastcells transduced by a viral vector with a BDNF gene insert. Toaccomplish this type of ex vivo gene transfer, Rejali et al. transducedguinea pig fibroblasts with an adenovirus with a BDNF gene cassetteinsert, and determined that these cells secreted BDNF and then attachedBDNF-secreting cells to the cochlear implant electrode via an agarosegel, and implanted the electrode in the scala tympani. Rejali et al.determined that the BDNF expressing electrodes were able to preservesignificantly more spiral ganglion neurons in the basal turns of thecochlea after 48 days of implantation when compared to controlelectrodes and demonstrated the feasibility of combining cochlearimplant therapy with ex vivo gene transfer for enhancing spiral ganglionneuron survival. Such a system may be applied to the CRISPR Cas systemof the present invention for delivery to the ear.

Mukherjea et al. (Antioxidants & Redox Signaling, Volume 13, Number 5,2010) document that knockdown of NOX3 using short interfering (si) RNAabrogated cisplatin ototoxicity, as evidenced by protection of OHCs fromdamage and reduced threshold shifts in auditory brainstem responses(ABRs). Different doses of siNOX3 (0.3, 0.6, and 0.9 μg) wereadministered to rats and NOX3 expression was evaluated by real timeRT-PCR. The lowest dose of NOX3 siRNA used (0.3 μg) did not show anyinhibition of NOX3 mRNA when compared to transtympanic administration ofscrambled siRNA or untreated cochleae. However, administration of thehigher doses of NOX3 siRNA (0.6 and 0.9 μg) reduced NOX3 expressioncompared to control scrambled siRNA. Such a system may be applied to theCRISPR Cas system of the present invention for transtympanicadministration with a dosage of about 2 mg to about 4 mg of CRISPR Casfor administration to a human.

Jung et al. (Molecular Therapy, vol. 21 no. 4, 834-841 April 2013)demonstrate that Hes5 levels in the utricle decreased after theapplication of siRNA and that the number of hair cells in these utricleswas significantly larger than following control treatment. The datasuggest that siRNA technology may be useful for inducing repair andregeneration in the inner ear and that the Notch signaling pathway is apotentially useful target for specific gene expression inhibition. Junget al. injected 8 μg of Hes5 siRNA in 2 μl volume, prepared by addingsterile normal saline to the lyophilized siRNA to a vestibularepithelium of the ear. Such a system may be applied to the CRISPR Cassystem of the present invention for administration to the vestibularepithelium of the ear with a dosage of about 1 to about 30 mg of CRISPRCas for administration to a human.

Pinyon J. L. et al. (Sci. Transl Med. 2014 Apr. 23; 6(233):233ra54. doi:10.1126/scitranslmed.3008177. Close-field electroporation gene deliveryusing the cochlear implant electrode array enhances the bionic ear.)reported their studies in guinea pigs which showed that neurotrophingene therapy integrated into the cochlear implant improved itsperformance by stimulating spiral ganglion neurite regeneration. Theauthors used the cochlear implant electrode array for novel“close-field” electroporation to transduce mesenchymal cells lining thecochlear perilymphatic canals with a naked complementary DNA geneconstruct driving expression of brain-derived neurotrophic factor (BDNF)and a green fluorescent protein (GFP) reporter. The focusing of electricfields by particular cochlear implant electrode configurations led tosurprisingly efficient gene delivery to adjacent mesenchymal cells. Theresulting BDNF expression stimulated regeneration of spiral ganglionneurites, which had atrophied 2 weeks after ototoxic treatment, in abilateral sensorineural deafness model. In this model, delivery of acontrol GFP-only vector failed to restore neuron structure, withatrophied neurons indistinguishable from unimplanted cochleae. With BDNFtherapy, the regenerated spiral ganglion neurites extended close to thecochlear implant electrodes, with localized ectopic branching. Thisneural remodeling enabled bipolar stimulation via the cochlear implantarray, with low stimulus thresholds and expanded dynamic range of thecochlear nerve, determined via electrically evoked auditory brainstemresponses. This development may broadly improve neural interfaces andextend molecular medicine applications.

Atkinson P. J. et al. (PLoS One 2014 Jul. 18; 9(7):e102077. doi:10.1371/journal.pone.0102077. eCollection 2014. Hair cell regenerationafter ATOH1 gene therapy in the cochlea of profoundly deaf adult guineapigs.) reported the results of a study aimed to promote the regenerationof sensory hair cells in the mature cochlea and their reconnection withauditory neurons through the introduction of ATOH1, a transcriptionfactor known to be necessary for hair cell development, and theintroduction of neurotrophic factors. Adenoviral vectors containingATOH1 alone, or with neurotrophin-3 and brain derived neurotrophicfactor were injected into the lower basal scala media of guinea pigcochleae four days post ototoxic deafening. Guinea pigs treated withATOH1 gene therapy, alone, had a significantly greater number of cellsexpressing hair cell markers compared to the contralateral non-treatedcochlea when examined 3 weeks post-treatment. This increase, however,did not result in a commensurate improvement in hearing thresholds, norwas there an increase in synaptic ribbons, as measured by CtBP2 punctaafter ATOH1 treatment alone, or when combined with neurotrophins.However, hair cell formation and synaptogenesis after co-treatment withATOH1 and neurotrophic factors remain inconclusive as viral transductionwas reduced due to the halving of viral titres when the samples werecombined. The authors concluded that collectively, these data suggeststhat, whilst ATOH1 alone can drive non-sensory cells towards an immaturesensory hair cell phenotype in the mature cochlea, this does not resultin functional improvements after aminoglycoside-induced deafness.

Deafness and Hearing Impairments Gene Therapy Description

One major cause of bearing and balance impairments is the loss of haircell within the human cochleae. The loss can be due to noise, ototoxicdamage, etc. Unfortunately, there is no evidence to support that newhair cells can be produced spontaneously in adult mammals includinghumans, and there is no method of reliably stimulating hair cellregeneration in mammalian

Cochleae after Birth

Nonetheless, recent reports have shown that overexpression of certaingene such as human ATOH1 can induce the production of new hair cells.ATOH1 is a basic helix-loop-helix (bHLH) transcription factor that hasbeen shown to be a key regulator in hair cell regeneration. Thus, themodulation of ATOH1 expression in vivo via epigenetic engineering usingCRISPR-Cas 9 system can be used as a potential therapeutic approach forhuman deafness or other types of hearing impairments. The majorchallenges for development of effective gene therapy for this type ofhearing diseases are: (a) Lack of easily designable genome engineering:This is addressed by the CRISPR-Cas9 technology. In particular, theability to create non-cleaving mutant version of the Cas9 protein,dCas9, that is capable of binding to target DNA sequence but notintroducing any DNA damage or modification. (b) Low efficiency of invivo delivery: This is improved by the small Cas9, SaCas9 fromStaphylococcus aureus, which can be readily packaged into a single AAVvector to express both the dCas9 protein, fusion effector, andcorresponding chimeric guide RNA(s). (c) Low efficiency of epigeneticmodulation due to necessity to apply multiple guides to manipulate asingle gene and thus the requirement of co-delivery of multiple viralvectors.

Applicants have solved the multiple-guide issue by the optimization ofdCas9 and the fusion effector, chimeric guide RNA design. In particular,multiple MS2 binding sites could be engineered into the chimeric RNAbackbone through tandem insertion. In this way, the epigeneticengineering is carried out by the tri-component complex consists ofdCas9, the modified chimeric guide RNA, and fusion efforts. The fusioneffectors harbor the MS2 protein and the epigenetic modifiers such asVP64, p65, KRAB, SID, or SID4X domains. Importantly, other RNA-proteininteractions can be explored as well in the same manner. Additionally,the multiple-vector issue is also improved by the introduction of smallSaCas9, that reduced the number of viral vectors required to perform theexperiments from (2 or 3 vectors in total) to just (1 or 2 vectors intotal).

Applicants genome engineering system using SaCas9 could be effectivepackaged into AAV or Ad viruses, and in particular can be used to modifyendogenous epigenetic state in mammalian cells in vivo, thereby modulatethe expression level of disease-relevant gene or genomic loci to executetherapeutic effects. The components of the system in a single-vectordesign is shown on FIG. 24, which shows design of dCas9-based epigeneticmodulation system (3 components of the system, dSaCas9, fusion effector,and sgRNA are shown). This system can be combined with delivery methodbased on Adeno-associated virus (AAV), Adeno viruses (Ad) or otherdelivery vehicles to modify the epigenetic state of cells in vivo.

An non-limiting example for use of dSaCas9 to stimulate ATOH1 expressionto treat deafness or hearing impairments is provided herein at Example6, and FIGS. 24 and 25.

Eyes

The present invention also contemplates delivering the CRISPR-Cas systemto one or both eyes.

In yet another aspect of the invention, the CRISPR-Cas system may beused to correct ocular defects that arise from several genetic mutationsfurther described in Genetic Diseases of the Eye, Second Edition, editedby Elias I. Traboulsi, Oxford University Press, 2012.

For administration to the eye, lentiviral vectors, in particular equineinfectious anemia viruses (EIAV) are particularly preferred.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285, Published online 21 Nov. 2005 in Wiley InterScience.DOI: 10.1002/jgm.845). The vectors are contemplated to havecytomegalovirus (CMV) promoter driving expression of the target gene.Intracameral, subretinal, intraocular and intravitreal injections areall contemplated (see, e.g., Balagaan, J Gene Med 2006; 8: 275-285,Published online 21 Nov. 2005 in Wiley InterScience. DOI:10.1002/jgm.845). Intraocular injections may be performed with the aidof an operating microscope. For subretinal and intravitreal injections,eyes may be prolapsed by gentle digital pressure and fundi visualisedusing a contact lens system consisting of a drop of a coupling mediumsolution on the cornea covered with a glass microscope slide coverslip.For subretinal injections, the tip of a 10-mm 34-gauge needle, mountedon a 5-μl Hamilton syringe may be advanced under direct visualisationthrough the superior equatorial sclera tangentially towards theposterior pole until the aperture of the needle was visible in thesubretinal space. Then, 2 μl of vector suspension may be injected toproduce a superior bullous retinal detachment, thus confirmingsubretinal vector administration. This approach creates a self-sealingsclerotomy allowing the vector suspension to be retained in thesubretinal space until it is absorbed by the RPE, usually within 48 h ofthe procedure. This procedure may be repeated in the inferior hemisphereto produce an inferior retinal detachment. This technique results in theexposure of approximately 70% of neurosensory retina and RPE to thevector suspension. For intravitreal injections, the needle tip may beadvanced through the sclera 1 mm posterior to the corneoscleral limbusand 2 μl of vector suspension injected into the vitreous cavity. Forintracameral injections, the needle tip may be advanced through acorneoscleral limbal paracentesis, directed towards the central cornea,and 2 μl of vector suspension may be injected. For intracameralinjections, the needle tip may be advanced through a corneosclerallimbal paracentesis, directed towards the central cornea, and 2 μl ofvector suspension may be injected. These vectors may be injected attitres of either 1.0-1.4×10¹⁰ or 1.0-1.4×10⁹ transducing units (TU)/ml.

In another embodiment, RetinoStat®, an equine infectious anemiavirus-based lentiviral gene therapy vector that expresses angiostaticproteins endostain and angiostatin that is delivered via a subretinalinjection for the treatment of the web form of age-related maculardegeneration is also contemplated (see, e.g., Binley et al., HUMAN GENETHERAPY 23:980-991 (September 2012)). Such a vector may be modified forthe CRISPR-Cas system of the present invention. Each eye may be treatedwith either RetinoStat® at a dose of 1.1×10⁵ transducing units per eye(TU/eye) in a total volume of 100 μl.

In another embodiment, an E1-, partial E3-, E4-deleted adenoviral vectormay be contemplated for delivery to the eye. Twenty-eight patients withadvanced neovascular age-related macular degeneration (AMD) were given asingle intravitreous injection of an E1-, partial E3-, E4-deletedadenoviral vector expressing human pigment ep-ithelium-derived factor(AdPEDF.ll) (see, e.g., Campochiaro et al., Human Gene Therapy17:167-176 (February 2006)). Doses ranging from 10⁶ to 10^(9.5) particleunits (PU) were investigated and there were no serious adverse eventsrelated to AdPEDF.ll and no dose-limiting toxicities (see, e.g.,Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006)).Adenoviral vector-mediated ocular gene transfer appears to be a viableapproach for the treatment of ocular disorders and could be applied tothe CRISPR Cas system.

In another embodiment, the sd-rxRNA® system of RXi Pharmaceuticals maybe used/and or adapted for delivering CRISPR Cas to the eye. In thissystem, a single intravitreal administration of 3 μg of sd-rxRNA resultsin sequence-specific reduction of PPIB mRNA levels for 14 days. The thesd-rxRNA® system may be applied to the CRISPR Cas system of the presentinvention, contemplating a dose of about 3 to 20 mg of CRISPRadministered to a human.

Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 April2011) describes adeno-associated virus (AAV) vectors to deliver an RNAinterference (RNAi)-based rhodopsin suppressor and a codon-modifiedrhodopsin replacement gene resistant to suppression due to nucleotidealterations at degenerate positions over the RNAi target site. Aninjection of either 6.0×10⁸ vp or 1.8×10¹⁰ vp AAV were subretinallyinjected into the eyes by Millington-Ward et al. The AAV vectors ofMillington-Ward et al. may be applied to the CRISPR Cas system of thepresent invention, contemplating a dose of about 2×10¹¹ to about 6×10¹³vp administered to a human.

Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also relates to invivo directed evolution to fashion an AAV vector that delivers wild-typeversions of defective genes throughout the retina after noninjuriousinjection into the eyes' vitreous humor. Dalkara describes a a 7merpeptide display library and an AAV library constructed by DNA shufflingof cap genes from AAV1, 2, 4, 5, 6, 8, and 9. The rcAAV libraries andrAAV vectors expressing GFP under a CAG or Rho promoter were packagedand and deoxyribonuclease-resistant genomic titers were obtained throughquantitative PCR. The libraries were pooled, and two rounds of evolutionwere performed, each consisting of initial library diversificationfollowed by three in vivo selection steps. In each such step, P30rho-GFP mice were intravitreally injected with 2 ml ofiodixanol-purified, phosphate-buffered saline (PBS)-dialyzed librarywith a genomic titer of about 1×10¹² vg/ml. The AAV vectors of Dalkaraet al. may be applied to the CRISPR Cas system of the present invention,contemplating a dose of about 1×10¹⁵ to about 1×10¹⁶ vg/ml administeredto a human.

In another embodiment, the rhodopsin gene may be targeted for thetreatment of retinitis pigmentosa (RP), wherein the system of US PatentPublication No. 20120204282 assigned to Sangamo BioSciences, Inc. may bemodified in accordance of the CRISPR Cas system of the presentinvention.

In another embodiment, the methods of US Patent Publication No.20130183282 assigned to Cellectis, which is directed to methods ofcleaving a target sequence from the human rhodopsin gene, may also bemodified to the CRISPR Cas system of the present invention.

US Patent Publication No. 20130202678 assigned to Academia Sinicarelates to methods for treating retinopathies and sight-threateningophthalmologic disorders relating to delivering of the Puf-A gene (whichis expressed in retinal ganglion and pigmented cells of eye tissues anddisplays a unique anti-apoptotic activity) to the sub-retinal orintravitreal space in the eye. In particular, desirable targets arezgc:193933, prdm1a, spata2, tex10, rbb4, ddx3, zp2.2, Blimp-1 and HtrA2,all of which may be targeted by the CRISPR Cas system of the presentinvention.

Wu (Cell Stem Cell, 13:659-62, 2013) designed a guide RNA that led Cas9to a single base pair mutation that causes cataracts in mice, where itinduced DNA cleavage. Then using either the other wild-type allele oroligos given to the zygotes repair mechanisms corrected the sequence ofthe broken allele and corrected the cataract-causing genetic defect inmutant mouse.

US Patent Publication No. 20120159653, describes use of zinc fingernucleases to genetically modify cells, animals and proteins associatedwith macular degeration (MD). Macular degeneration (MD) is the primarycause of visual impairment in the elderly, but is also a hallmarksymptom of childhood diseases such as Stargardt disease, Sorsby fundus,and fatal childhood neurodegenerative diseases, with an age of onset asyoung as infancy. Macular degeneration results in a loss of vision inthe center of the visual field (the macula) because of damage to theretina. Currently existing animal models do not recapitulate majorhallmarks of the disease as it is observed in humans. The availableanimal models comprising mutant genes encoding proteins associated withMD also produce highly variable phenotypes, making translations to humandisease and therapy development problematic.

One aspect of US Patent Publication No. 20120159653 relates to editingof any chromosomal sequences that encode proteins associated with MDwhich may be applied to the CRISPR Cas system of the present invention.The proteins associated with MD are typically selected based on anexperimental association of the protein associated with MD to an MDdisorder. For example, the production rate or circulating concentrationof a protein associated with MD may be elevated or depressed in apopulation having an MD disorder relative to a population lacking the MDdisorder. Differences in protein levels may be assessed using proteomictechniques including but not limited to Western blot,immunohistochemical staining, enzyme linked immunosorbent assay (ELISA),and mass spectrometry. Alternatively, the proteins associated with MDmay be identified by obtaining gene expression profiles of the genesencoding the proteins using genomic techniques including but not limitedto DNA microarray analysis, serial analysis of gene expression (SAGE),and quantitative real-time polymerase chain reaction (Q-PCR).

By way of non-limiting example, proteins associated with MD include butare not limited to the following proteins: (ABCA4) ATP-binding cassette,sub-family A (ABC1), member 4 ACHM1 achromatopsia (rod monochromacy) 1ApoE Apolipoprotein E (ApoE) C1QTNF5 (CTRP5) C1q and tumor necrosisfactor related protein 5 (C1QTNF5) C2 Complement component 2 (C2) C3Complement components (C3) CCL2 Chemokine (C-C motif) Ligand 2 (CCL2)CCR2 Chemokine (C-C motif) receptor 2 (CCR2) CD36 Cluster ofDifferentiation 36 CFB Complement factor B CFH Complement factor CFH HCFHR1 complement factor H-related 1 CFHR3 complement factor H-related 3CNGB3 cyclic nucleotide gated channel beta 3 CP ceruloplasmin (CP) CRP Creactive protein (CRP) CST3 cystatin C or cystatin 3 (CST3) CTSDCathepsin D (CTSD) CX3CR1 chemokine (C-X3-C motif) receptor 1 ELOVL4Elongation of very long chain fatty acids 4 ERCC6 excision repaircross-complementing rodent repair deficiency, complementation group 6FBLN5 Fibulin-5 FBLN5 Fibulin 5 FBLN6 Fibulin 6 FSCN2 fascin (FSCN2)HMCN1 Hemicentrin 1 HMCN1 hemicentin 1 HTRA1 HtrA serine peptidase 1(HTRA1) HTRA1 HtrA serine peptidase 1 IL-6 Interleukin 6 IL-8Interleukin 8 LOC387715 Hypothetical protein PLEKHA1 Pleckstrin homologydomain-containing family A member 1 (PLEKHA1) PROM1 Prominin 1(PROM1 orCD133) PRPH2 Peripherin-2 RPGR retinitis pigmentosa GTPase regulatorSERPING1 serpin peptidase inhibitor, clade G, member 1 (C1-inhibitor)TCOF1 Treacle TIMP3 Metalloproteinase inhibitor 3 (TIMP3) TLR3 Toll-likereceptor 3.

The identity of the protein associated with MD whose chromosomalsequence is edited can and will vary. In preferred embodiments, theproteins associated with MD whose chromosomal sequence is edited may bethe ATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4)encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded bythe APOE gene, the chemokine (C-C motif) Ligand 2 protein (CCL2) encodedby the CCL2 gene, the chemokine (C-C motif) receptor 2 protein (CCR2)encoded by the CCR2 gene, the ceruloplasmin protein (CP) encoded by theCP gene, the cathepsin D protein (CTSD) encoded by the CTSD gene, or themetalloproteinase inhibitor 3 protein (TIMP3) encoded by the TIMP3 gene.In an exemplary embodiment, the genetically modified animal is a rat,and the edited chromosomal sequence encoding the protein associated withMD may be: (ABCA4) ATP-binding cassette, NM_000350 sub-family A (ABC1),member 4 APOE Apolipoprotein E NM_138828 (APOE) CCL2 Chemokine (C-CNM_031530 motif) Ligand 2 (CCL2) CCR2 Chemokine (C-C NM_021866 motif)receptor 2 (CCR2) CP ceruloplasmin (CP) NM_012532 CTSD Cathepsin D(CTSD) NM_134334 TIMP3 Metalloproteinase NM_012886 inhibitor 3 (TIMP3)The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7 or more disruptedchromosomal sequences encoding a protein associated with MD and zero, 1,2, 3, 4, 5, 6, 7 or more chromosomally integrated sequences encoding thedisrupted protein associated with MD.

The edited or integrated chromosomal sequence may be modified to encodean altered protein associated with MD. Several mutations in MD-relatedchromosomal sequences have been associated with MD. Non-limitingexamples of mutations in chromosomal sequences associated with MDinclude those that may cause MD including in the ABCR protein, E471K(i.e. glutamate at position 471 is changed to lysine), R1129L (i.e.arginine at position 1129 is changed to leucine), T1428M (i.e. threonineat position 1428 is changed to methionine), R1517S (i.e. arginine atposition 1517 is changed to serine), I1562T (i.e. isoleucine at position1562 is changed to threonine), and G1578R (i.e. glycine at position 1578is changed to arginine); in the CCR2 protein, V64I (i.e. valine atposition 192 is changed to isoleucine); in CP protein, G969B (i.e.glycine at position 969 is changed to asparagine or aspartate); in TIMP3protein, S156C (i.e. serine at position 156 is changed to cysteine),G166C (i.e. glycine at position 166 is changed to cysteine), G167C (i.e.glycine at position 167 is changed to cysteine), Y168C (i.e. tyrosine atposition 168 is changed to cysteine), S170C (i.e. serine at position 170is changed to cysteine), Y172C (i.e. tyrosine at position 172 is changedto cysteine) and S181C (i.e. serine at position 181 is changed tocysteine). Other associations of genetic variants in MD-associated genesand disease are known in the art.

Ocular Disease Gene Therapy

There are many types of hereditary retina disease that have been mappedextensively for their genetic basis and thus provide good avenue foremploying genome engineering technology to develop effective genetherapy to treat these conditions in human patients. Based on thefeasibility, a list of ocular diseases is shown on the table below withannotations on their genetics and mode of inheritance.

Disease List and Annotations

Ocular diseases Causal genomic loci in human genome Mode of inheritanceStargardt Disease and ABCA4 & ELOVL4 Autosomal Recessive and RetinalDegeneration Autosomal Dominant Achromatopsia CNGA3(exon 8) & CNGB3(Exon10) Autosomal Recessive CNGA3, CNGB3, CNNM4, GNAT2, Autosomal Recessive& X-linked KCNV2, NBAS, OPN1LW, PDE6C, PDE6H & RPGR Bardet-BiedlSyndrome ARL6, BBS1, BBS2, BBS4, BBS5, BBS7, Autosomal Recessive BBS9,BBS10, BBS12, CEP290, INPP5E, LZTFL1, MKS1, MKKS, SDCCAG8, TRIM32 & TTC8Best Disease BEST1 Autosomal Dominant Blue Cone OPNL1W X-LinkedMonochromacy Choroideremia CHM X-Linked Cone-Rod Dystrophy CRX,GUCA1A(Leu151Phe) & GUCY2D Autosomal Dominant (Exon 13) ADAM9, AIPL1,C21ORF2, C8ORF37, Autosomal Dominant, Autosomal CACNA1F, CACNA2D4,CDHR1, Recessive & X-Linked CERKL, CNGB3, CNNM4, CRX, GUCA1A, GUCY2D,KCNV2, PDE6C, PDE6H, PITPNM3, PROM1, PRPH2, RAP28, RAX2, RDH5, RIMS1,RPGR, RPGRIP1 & UNC119 Congenital Stationary CACNA1F, GRM6, PDE6B &TRPM1 Autosomal Dominant, Autosomal Night Blindness Recessive & X-LinkedCABP4, CACNA1F, GNAT1, GPR179, GRK1, GRM6, LRIT3, NYX, PDE6B, RDH5, RHO,SAG, SLC24A1, TRPM1 Corneal Dystrophy- TGFBI (Exons 4 & 11-14) AutosomalDominant Stromal Enhanced S-Cone NR2E3 (Exons 2-8) Autosomal RecessiveSyndrome Juvenile Open Angle MYOC Autosomal Dominant Glaucoma or PrimaryOpen Angle Glaucoma Juvenile X-Linked RS1 X-Linked Retinoschisis LeberCongenital AIPL1, CEP290, CRB1, CRX, GUCY2D, Autosomal RecessiveAmaurosis (LCA) IQCB1, LCA5, LRAT, NMNAT1, RD3, RDH12, RPE65, RPGRIP1,SPATA7, TULP1 Malattia Leventinese EFEMP1 (Arg345Trp mutation) AutosomalDominant Norrie Disease or X- NDP X-Linked Linked Familial ExudativeVitreoretinopathy (XL- FEVR) Pattern Dystrophy RDS Autosomal DominantRetinitis Pigmentosa C1QTNF5, IMPDH1, NR2E3, PRPF3, Autosomal DominantPRPF31, PRPF8, RDH12, RDS, RHO, RP1, RP9, SNRNP200, TOPORS ABCA4,CC2D2A, CERKL, CLRN1, Autosomal Recessive CNGA1, CRB1, DHDDS, EYS,FAM161A, FLVCR1, IDH3B, IMPG2, LRAT, MAK, NR2E3, NRL, PDE6A, PDE6B,PDE6G, PROM1, RBP3, RDH12, RGR, RLBP1, RPE65, SAG, TTPA, TULP1, USH2A,ZNF513 RP2, RPGR X-Linked Sorsby Dystrophy TIMP3 (Exons 1 & 5) AutosomalDominant Usher Syndrome ABHD12, CDH23, CIB2, CLRN1, Autosomal RecessiveDFNB31, GPR98, HARS, MYO7A, PCDH15, USH1C, USH1G & USH2A Aniridia PAX6Autosomal Dominant Dominant Optic Atrophy OPA1 Autosomal Dominant

The major challenges for development of effective gene therapy forocular diseases are: (a) lack of easily designable genome engineering;(b) low efficiency of in vivo delivery; and (c) low efficiency of HDRdue to co-delivery of multiple viral vectors. Applicants have shown thatthe CRISPR-Cas9 technology effectively addresses and provides solutionsto these challenges. Applicants have shown that the challenges of lowefficiency of in vivo delivery and low efficiency of HDR and co-deliveryis solved by the small Cas9, SaCas9 from Staphylococcus aureus, whichcan be readily packaged into a single Adeno-associated virus (AAV)vector to express both the Cas9 protein and its corresponding sgRNA(s).Further, Applicants have shown that introduction of small SaCas9, hasreduced the number of viral vectors required to performhomology-directed repair (HDR) from 3 vectors to 2 vectors.

Modifying endogenous genome sequence in mammalian cells in vivo usingSaCas9-based CRISPR-Cas genome engineering system:

-   Applicants have shown that a genome engineering system using SaCas9    can be effectively packaged into AAV, and in particular can be used    to modify endogenous genome sequence in mammalian cells in vivo.-   The basic features of Applicants' SaCas9 system is shown in FIG. 15.    This system can be combined with delivery methods based on    Adeno-associated virus (AAV) to edit post-mitotic cells in vivo and    is effective for a number of cell types in human retina when    combined with specific delivery vehicles. In the case of retina    disease therapy, two delivery routes are employed: intravitreal AAV    injection, where AAV is injected in the vitreous humor of the eye,    can be used to targets retinal ganglion cells and Muller glial    cells, or to systain long-term expression of the transgene within    ocular cells. On the other hand, subretinal AAV injection, where a    small amount of fluid is injected underneath the retina, efficiently    targets photoreceptors and retinal pigment epithelium (RPE) cells.

AAV serotype 2 and 8 (AAV2 and AAV8) are the most effective serotypesthat can be used for the delivery using the intravitral route forganglion and Muller cells. AAV serotype 1, 2, 5, 7, 8, DJ can be used todeliver transgene into the photoreceptor and RPE cells via thesubretinal injection procedure. The detailed model for gene therapyusing this system is shown in FIG. 16.

It will be readily appreciated that in vivo therapeutic genomeengineering approach described herein, illustrated in FIG. 16, andexemplified in Examples 2-5, can be employed to correct disease-causingmutations or other types of genomic abnormalities in the ocular system.The protocol can be summarized into the following steps: (i) in vitrotarget and HDR template validation; (ii) virus production; (iii) viruspurification; and (iv) ocular injection.

In Vivo Therapeutic Genome Engineering for Retinitis Pigmentosa

Retinitis Pigmentosa (RP) is a hereditary ocular disorder that can leadto vision impairement and in some cases blindness. It is a type ofdegenerative eye disease that is often caused by missense mutations ingenes involved in the function or regulation of photoreceptors cells orretinal pigment epithelium (RPE) cells in human eyes. RP is one of themost common forms of inherited retinal degeneration.

A key gene in the molecular genetics of RP is the rhodopsin gene (RHO).RHO gene encodes a principal protein of photoreceptor outer segments.Studies show that mutations in this gene are responsible forapproximately 25% of autosomal dominant forms of RP.

One example of RHO mutation that causes RP is nucleotide substitution atcodon 23, CCC to CAC, which encoding the amino acid substitution ofhistidine for phenylalanine at position 23 of the RHO gene, or RHO(P23H). The P23H mutation is one of the most common causes of autosomaldominant retinitis pigmentosa (FIG. 18A). The phenotype in heterozygouspatient is predominantly retinopathy and progressive retinaldegeneration. Patients homozygous for this disease exhibit a more severephenotype. It has been observed that glycosylation of the mutant P23Hprotein is severely diminished. In general, patients may experiencedefective light to dark, dark to light adaptation or nyctalopia, as theresult of the degeneration of the peripheral visual field. Centralvision loss is also observed in some cases. RP can be non-syndromic, orsyndromic with deafness, ataxia, etc.

List of Other RP Disease Mutations Genomic Abnormalities

Disease Mutated Genes Mode of inheritance Retinitis C1QTNF5, IMPDH1,NR2E3, PRPF3, PRPF31, Autosomal Dominant Pigmentosa PRPF8, RDH12, RDS,RHO, RP1, RP9, SNRNP200, TOPORS ABCA4, CC2D2A, CERKL, CLRN1, CNGA1,Autosomal Recessive CRB1, DHDDS, EYS, FAM161A, FLVCR1, IDH3B, IMPG2,LRAT, MAK, NR2E3, NRL, PDE6A, PDE6B, PDE6G, PROM1, RBP3, RDH12, RGR,RLBP1, RPE65, SAG, TTPA, TULP1, USH2A, ZNF513 RP2, RPGR X-Linked

As noted above, it will be readily appreciated that Applicants'exemplary in vivo therapeutic genome engineering approach describedherein and shown in FIG. 5 can be employed to correct disease-causingmutations or other types of genomic abnormalities in the ocular system.Applicants' genome engineering approach for retinitis pigmentosa genetherapy targeting the RHO gene is discussed in Examples 2 and 3 herein.

In Vivo Therapeutic Genome Engineering for Achromatopsia

Achromatopsia (ACHM) is a medical condition that is described by theinability of the patient to perceive color, maintain normal visualacuity at high light levels (i.e. exterior daylight). Although it canrefer to acquired conditions, it typically refers to the autosomalrecessive congenital color vision condition. The condition can alsomanifest as an incomplete form, defined as dyschromatopsia. Theestimated occurrence is around 1 in 33,000 people in the generalpopulation.

There are five major symptoms that are associated with ACHM, namelyachromatopia, amblyopia, hemeralopia, nystagmus, and iris operatingabnormalities.

The key gene in the molecular genetics of ACHM is the cone cell cyclicnucleotide-gated ion channel genes ACNGA3, CNGB3, and transducing geneGNAT2. Mutations in these genes will lead to malfunction of the retinalphototransduction pathway. Specifically, this type of congenital ACHM isthought to result from the inability of cone cells to properly respondto light input by hyperpolarizing. Achromatopsia caused by CNGA3mutation is categorized as ACHM2, CNGB3 mutation as ACHM3, and GNAT2 asACHM4. These are the major types of ACHM, while some minority of casesare caused by mutation of gene PDE6C and other genes, called ACHM5.

Applicants' genome engineering approach for ACHM gene therapy targetingthe CNGA3 and CNGB3 mutations is described herein in Example 4.

For CNGA3 (ACHM2), there are four major mutations, arg277 to cys(R277C), arg283 to trp (R283W), arg436 to trp (R435W), and phe547 to leu(F547L). These disease-causing mutations accounted for 41.8% of all thedetected mutations (from the report in Wissinger et al. 2001, Am. J.Hum. Genet.). Here Applicants select the first and second mutation(R277C and R283W) as example. Because their close proximity, these twomutations can be corrected with the same strategy, constructs, viralvector sets, and procedure.

For CNGB3 (ACHM3), the 1148delC mutation is a prevalent form ofdisease-causing mutation and has been reported to account for 75% ofpatients (Wiszniewski et al. 2007). And the correction of this mutationthrough Cas9-mediated genome engineering approach with a HR templatevector will be able to rescue the disease phenotype.

List of Other Achromatopsia Disease Mutations/Genomic Abnormalities

Disease Mutated Genes Mode of inheritance Achromatopsia CNGA3, CNGB3,CNNM4, Autosomal Recessive GNAT2, KCNV2, NBAS, OPN1LW, PDE6C, PDE6H &RPGR

In Vivo Therapeutic Genome Engineering for Age Related MacularDegeneration

Age-related macular degeneration (AMD or ARMD) is a medical conditionthat usually affects older adults and results in a loss of vision in thecenter of the visual field because of damage to the retina. It occurs in“dry” and “wet” forms. It is a major cause of blindness and visualimpairment in adults aged with 50.

There are two types of ARMD, the wet and the dry forms. The wet orexudative form of ARMD is characterized by angiogenesis from the choroidbehind the retina. The new vessels are fragile and can result in bloodand protein leakage below the macula. Bleeding, leaking, and scarringfrom these blood vessels eventually cause irreversible damage to thephotoreceptors and thus rapid vision loss if left untreated. The retinacan become detached because of the growing blood vessels.

In the dry or nonexudative form, cellular debris called drusenaccumulates between the retina and the choroid, and this affect thevision of the patients and can ultimately cause retina detachment aswell.

Molecular Target for Treating Age-Related Macular Degeneration

The most relevant form of ARMD that can potentially be treated with genetherapy is the ‘wet’ form of ARMD. In this case, the proliferation ofabnormal blood vessels in the retina is stimulated by vascularendothelial growth factor (VEGF), or the genomic locus VEGFA in humangenome. Hence, methods that can repress VEGF expression or inhibit itsactivity can stop, or in some cases, reverse the growth of bloodvessels. This is a promising molecular approach to treat this type ofARMD effectively in human patients.

An exemplary non-limiting genome engineering approach for gene therapyfor treating age-related macular degeneration is exemplified herein atExample 5 and FIG. 23A-B.

Heart

The present invention also contemplates delivering the CRISPR-Cas systemto the heart. For the heart, a myocardium tropic adena-associated virus(AAVM) is preferred, in particular AAVM41 which showed preferential genetransfer in the heart (see, e.g., Lin-Yanga et al., PNAS, Mar. 10, 2009,vol. 106, no. 10). Administration may be systemic or local. A dosage ofabout 1-10×10¹⁴ vector genomes are contemplated for systemicadministration. See also, e.g., Eulalio et al. (2012) Nature 492: 376and Somasuntharam et al. (2013) Biomaterials 34: 7790.

For example, US Patent Publication No. 20110023139, describes use ofzinc finger nucleases to genetically modify cells, animals and proteinsassociated with cardiovascular disease. Cardiovascular diseasesgenerally include high blood pressure, heart attacks, heart failure, andstroke and TIA. Any chromosomal sequence involved in cardiovasculardisease or the protein encoded by any chromosomal sequence involved incardiovascular disease may be utilized in the methods described in thisdisclosure. The cardiovascular-related proteins are typically selectedbased on an experimental association of the cardiovascular-relatedprotein to the development of cardiovascular disease. For example, theproduction rate or circulating concentration of a cardiovascular-relatedprotein may be elevated or depressed in a population having acardiovascular disorder relative to a population lacking thecardiovascular disorder. Differences in protein levels may be assessedusing proteomic techniques including but not limited to Western blot,immunohistochemical staining, enzyme linked immunosorbent assay (ELISA),and mass spectrometry. Alternatively, the cardiovascular-relatedproteins may be identified by obtaining gene expression profiles of thegenes encoding the proteins using genomic techniques including but notlimited to DNA microarray analysis, serial analysis of gene expression(SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

By way of example, the chromosomal sequence may comprise, but is notlimited to, ILIB (interleukin 1, beta), XDH (xanthine dehydrogenase),TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin)synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1),ABCG8 (ATP-binding cassette, sub-family G (WHITE), member 8), CTSK(cathepsin K), PTGIR (prostaglandin 12 (prostacyclin) receptor (IP)),KCNJ11 (potassium inwardly-rectifying channel, subfamily J, member 11),INS (insulin), CRP (C-reactive protein, pentraxin-related), PDGFRB(platelet-derived growth factor receptor, beta polypeptide), CCNA2(cyclin A2), PDGFB (platelet-derived growth factor beta polypeptide(simian sarcoma viral (v-sis) oncogene homolog)), KCNJ5 (potassiuminwardly-rectifying channel, subfamily J, member 5), KCNN3 (potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 3), CAPN10 (calpain 10), PTGES (prostaglandin E synthase), ADRA2B(adrenergic, alpha-2B-, receptor), ABCG5 (ATP-binding cassette,sub-family G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAPN5 (calpain5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C(mex-3 homolog C (C. elegans)), ACE angiotensin I converting enzyme(peptidyl-dipeptidase A) 1), TNF (tumor necrosis factor (TNFsuperfamily, member 2)), IL6 (interleukin 6 (interferon, beta 2)), STN(statin), SERPINEl (serpin peptidase inhibitor, clade E (nexin,plasminogen activator inhibitor type 1), member 1), ALB (albumin),ADIPOQ (adiponectin, CIQ and collagen domain containing), APOB(apolipoprotein B (including Ag(x) antigen)), APOE (apolipoprotein E),LEP (leptin), MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)),APOA1 (apolipoprotein A-I), EDN1 (endothelin 1), NPPB (natriureticpeptide precursor B), NOS3 (nitric oxide synthase 3 (endothelial cell)),PPARG (peroxisome proliferator-activated receptor gamma), PLAT(plasminogen activator, tissue), PTGS2 (prostaglandin-endoperoxidesynthase 2 (prostaglandin G/H synthase and cyclooxygenase)), CETP(cholesteryl ester transfer protein, plasma), AGTR1 (angiotensin IIreceptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme Areductase), IGF1 (insulin-like growth factor 1 (somatomedin C)), SELE(selectin E), REN (renin), PPARA (peroxisome proliferator-activatedreceptor alpha), PON1 (paraoxonase 1), KNG1 (kininogen 1), CCL2(chemokine (C-C motif) ligand 2), LPL (lipoprotein lipase), VWF (vonWillebrand factor), F2 (coagulation factor II (thrombin)), ICAM1(intercellular adhesion molecule 1), TGFB1 (transforming growth factor,beta 1), NPPA (natriuretic peptide precursor A), IL10 (interleukin 10),EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM1(vascular cell adhesion molecule 1), IFNG (interferon, gamma), LPA(lipoprotein, Lp(a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1),MAPK1 (mitogen-activated protein kinase 1), HP (haptoglobin), F3(coagulation factor III (thromboplastin, tissue factor)), CST3 (cystatinC), COG2 (component of oligomeric golgi complex 2), MMP9 (matrixmetallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IVcollagenase)), SERPINC1 (serpin peptidase inhibitor, clade C(antithrombin), member 1), F8 (coagulation factor VIII, procoagulantcomponent), HMOX1 (heme oxygenase (decycling) 1), APOC3 (apolipoproteinC-III), IL8 (interleukin 8), PROK1 (prokineticin 1), CBS(cystathionine-beta-synthase), NOS2 (nitric oxide synthase 2,inducible), TLR4 (toll-like receptor 4), SELP (selectin P (granulemembrane protein 140 kDa, antigen CD62)), ABCA1 (ATP-binding cassette,sub-family A (ABC1), member 1), AGT (angiotensinogen (serpin peptidaseinhibitor, clade A, member 8)), LDLR (low density lipoprotein receptor),GPT (glutamic-pyruvate transaminase (alanine aminotransferase)), VEGFA(vascular endothelial growth factor A), NR3C2 (nuclear receptorsubfamily 3, group C, member 2), IL18 (interleukin 18(interferon-gamma-inducing factor)), NOS1 (nitric oxide synthase 1(neuronal)), NR3C1 (nuclear receptor subfamily 3, group C, member 1(glucocorticoid receptor)), FGB (fibrinogen beta chain), HGF (hepatocytegrowth factor (hepapoietin A, scatter factor)), IL1A (interleukin 1,alpha), RETN (resistin), AKT1 (v-akt murine thymoma viral oncogenehomolog 1), LIPC (lipase, hepatic), HSPD1 (heat shock 60 kDa protein 1(chaperonin)), MAPK14 (mitogen-activated protein kinase 14), SPP1(secreted phosphoprotein 1), ITGB3 (integrin, beta 3 (plateletglycoprotein 111a, antigen CD61)), CAT (catalase), UTS2 (urotensin 2),THBD (thrombomodulin), F10 (coagulation factor X), CP (ceruloplasmin(ferroxidase)), TNFRSF11B (tumor necrosis factor receptor superfamily,member 11b), EDNRA (endothelin receptor type A), EGFR (epidermal growthfactor receptor (erythroblastic leukemia viral (v-erb-b) oncogenehomolog, avian)), MMP2 (matrix metallopeptidase 2 (gelatinase A, 72 kDagelatinase, 72 kDa type IV collagenase)), PLG (plasminogen), NPY(neuropeptide Y), RHOD (ras homolog gene family, member D), MAPK8(mitogen-activated protein kinase 8), MYC (v-myc myelocytomatosis viraloncogene homolog (avian)), FN1 (fibronectin 1), CMA1 (chymase 1, mastcell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotidebinding protein (G protein), beta polypeptide 3), ADRB2 (adrenergic,beta-2-, receptor, surface), APOA5 (apolipoprotein A-V), SOD2(superoxide dismutase 2, mitochondrial), F5 (coagulation factor V(proaccelerin, labile factor)), VDR (vitamin D (1,25-dihydroxyvitaminD3) receptor), ALOX5 (arachidonate 5-lipoxygenase), HLA-DRB1 (majorhistocompatibility complex, class II, DR beta 1), PARP1 (poly(ADP-ribose) polymerase 1), CD40LG (CD40 ligand), PON2 (paraoxonase 2),AGER (advanced glycosylation end product-specific receptor), IRS1(insulin receptor substrate 1), PTGS1 (prostaglandin-endoperoxidesynthase 1 (prostaglandin G/H synthase and cyclooxygenase)), ECE1(endothelin converting enzyme 1), F7 (coagulation factor VII (serumprothrombin conversion accelerator)), URN (interleukin 1 receptorantagonist), EPHX2 (epoxide hydrolase 2, cytoplasmic), IGFBP1(insulin-like growth factor binding protein 1), MAPK10(mitogen-activated protein kinase 10), FAS (Fas (TNF receptorsuperfamily, member 6)), ABCB1 (ATP-binding cassette, sub-family B(MDR/TAP), member 1), JUN (jun oncogene), IGFBP3 (insulin-like growthfactor binding protein 3), CD14 (CD14 molecule), PDE5A(phosphodiesterase 5A, cGMP-specific), AGTR2 (angiotensin II receptor,type 2), CD40 (CD40 molecule, TNF receptor superfamily member 5), LCAT(lecithin-cholesterol acyltransferase), CCR5 (chemokine (C-C motif)receptor 5), MMP1 (matrix metallopeptidase 1 (interstitialcollagenase)), TIMP1 (TIMP metallopeptidase inhibitor 1), ADM(adrenomedullin), DYT10 (dystonia 10), STAT3 (signal transducer andactivator of transcription 3 (acute-phase response factor)), MMP3(matrix metallopeptidase 3 (stromelysin 1, progelatinase)), ELN(elastin), USFI (upstream transcription factor 1), CFH (complementfactor H), HSPA4 (heat shock 70 kDa protein 4), MMP12 (matrixmetallopeptidase 12 (macrophage elastase)), MME (membranemetallo-endopeptidase), F2R (coagulation factor II (thrombin) receptor),SELL (selectin L), CTSB (cathepsin B), ANXA5 (annexin A5), ADRB1(adrenergic, beta-1-, receptor), CYBA (cytochrome b-245, alphapolypeptide), FGA (fibrinogen alpha chain), GGT1(gamma-glutamyltransferase 1), LIPG (lipase, endothelial), HIF1A(hypoxia inducible factor 1, alpha subunit (basic helix-loop-helixtranscription factor)), CXCR4 (chemokine (C-X-C motif) receptor 4), PROC(protein C (inactivator of coagulation factors Va and VIIIa)), SCARBI(scavenger receptor class B, member 1), CD79A (CD79a molecule,immunoglobulin-associated alpha), PLTP (phospholipid transfer protein),ADD1 (adducin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serumamyloid A1), KCNH2 (potassium voltage-gated channel, subfamily H(eag-related), member 2), DPP4 (dipeptidyl-peptidase 4), G6PD(glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptorA/guanylate cyclase A (atrionatriuretic peptide receptor A)), VTN(vitronectin), KIAA0101 (KIAA0101), FOS (FBJ murine osteosarcoma viraloncogene homolog), TLR2 (toll-like receptor 2), PPIG (peptidylprolylisomerase G (cyclophilin G)), ILiR1 (interleukin 1 receptor, type I), AR(androgen receptor), CYP1A1 (cytochrome P450, family 1, subfamily A,polypeptide 1), SERPINAl (serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 1), MTR(5-methyltetrahydrofolate-homocysteine methyltransferase), RBP4 (retinolbinding protein 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A(cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)),FGF2 (fibroblast growth factor 2 (basic)), EDNRB (endothelin receptortype B), ITGA2 (integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2receptor)), CABIN1 (calcineurin binding protein 1), SHBG (sexhormone-binding globulin), HMGB1 (high-mobility group box 1), HSP90B2P(heat shock protein 90 kDa beta (Grp94), member 2 (pseudogene)), CYP3A4(cytochrome P450, family 3, subfamily A, polypeptide 4), GJA1 (gapjunction protein, alpha 1, 43 kDa), CAV1 (caveolin 1, caveolae protein,22 kDa), ESR2 (estrogen receptor 2 (ER beta)), LTA (lymphotoxin alpha(TNF superfamily, member 1)), GDF15 (growth differentiation factor 15),BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450,family 2, subfamily D, polypeptide 6), NGF (nerve growth factor (betapolypeptide)), SP1 (Sp1 transcription factor), TGIF1 (TGFB-inducedfactor homeobox 1), SRC (v-src sarcoma (Schmidt-Ruppin A-2) viraloncogene homolog (avian)), EGF (epidermal growth factor(beta-urogastrone)), PIK3CG (phosphoinositide-3-kinase, catalytic, gammapolypeptide), HLA-A (major histocompatibility complex, class I, A),KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1),CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (cholinekinase alpha), BEST1 (bestrophin 1), APP (amyloid beta (A4) precursorprotein), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombospondinreceptor)), PRKABI (protein kinase, AMP-activated, beta 1 non-catalyticsubunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7family, member A1), CX3CR1 (chemokine (C-X3-C motif) receptor 1), TH(tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone1), TF (transferrin), HFE (hemochromatosis), IL17A (interleukin 17A),PTEN (phosphatase and tensin homolog), GSTM1 (glutathione S-transferasemu 1), DMD (dystrophin), GATA4 (GATA binding protein 4), F13A1(coagulation factor XIII, A1 polypeptide), TTR (transthyretin), FABP4(fatty acid binding protein 4, adipocyte), PON3 (paraoxonase 3), APOC1(apolipoprotein C-I), INSR (insulin receptor), TNFRSF1B (tumor necrosisfactor receptor superfamily, member 1B), HTR2A (5-hydroxytryptamine(serotonin) receptor 2A), CSF3 (colony stimulating factor 3(granulocyte)), CYP2C9 (cytochrome P450, family 2, subfamily C,polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450, family 11,subfamily B, polypeptide 2), PTH (parathyroid hormone), CSF2 (colonystimulating factor 2 (granulocyte-macrophage)), KDR (kinase insertdomain receptor (a type III receptor tyrosine kinase)), PLA2G2A(phospholipase A2, group IIA (platelets, synovial fluid)), B2M(beta-2-microglobulin), THBSI (thrombospondin 1), GCG (glucagon), RHOA(ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2family (mitochondrial)), TCF7L2 (transcription factor 7-like 2 (T-cellspecific, HMG-box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclearfactor (erythroid-derived 2)-like 2), NOTCH1 (Notch homolog 1,translocation-associated (Drosophila)), UGT1A1 (UDPglucuronosyltransferase 1 family, polypeptide A1), IFNA1 (interferon,alpha 1), PPARD (peroxisome proliferator-activated receptor delta),SIRT1 (sirtuin (silent mating type information regulation 2 homolog) 1(S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1(luteinizing-releasing hormone)), PAPPA (pregnancy-associated plasmaprotein A, pappalysin 1), ARR3 (arrestin 3, retinal (X-arrestin)), NPPC(natriuretic peptide precursor C), AHSP (alpha hemoglobin stabilizingprotein), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13),MTOR (mechanistic target of rapamycin (serine/threonine kinase)), ITGB2(integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)),GSTT1 (glutathione S-transferase theta 1), IL6ST (interleukin 6 signaltransducer (gp130, oncostatin M receptor)), CPB2 (carboxypeptidase B2(plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute carrierfamily 6 (neurotransmitter transporter, serotonin), member 4), PLA2G6(phospholipase A2, group VI (cytosolic, calcium-independent)), TNFSF11(tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solutecarrier family 8 (sodium/calcium exchanger), member 1), F2RL1(coagulation factor II (thrombin) receptor-like 1), AKR1A1 (aldo-ketoreductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehydedehydrogenase 9 family, member A1), BGLAP (bone gamma-carboxyglutamate(gla) protein), MTTP (microsomal triglyceride transfer protein), MTRR(5-methyltetrahydrofolate-homocysteine methyltransferase reductase),SULT1A3 (sulfotransferase family, cytosolic, 1A, phenol-preferring,member 3), RAGE (renal tumor antigen), C4B (complement component 4B(Chido blood group), P2RY12 (purinergic receptor P2Y, G-protein coupled,12), RNLS (renalase, FAD-dependent amine oxidase), CREB (cAMP responsiveelement binding protein 1), POMC (proopiomelanocortin), RAC1(ras-related C3 botulinum toxin substrate 1 (rho family, small GTPbinding protein Rac1)), LMNA (lamin NC), CD59 (CD59 molecule, complementregulatory protein), SCN5A (sodium channel, voltage-gated, type V, alphasubunit), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide1), MIF (macrophage migration inhibitory factor(glycosylation-inhibiting factor)), MMP13 (matrix metallopeptidase 13(collagenase 3)), TIMP2 (TIMP metallopeptidase inhibitor 2), CYP19A1(cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2(cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22(protein tyrosine phosphatase, non-receptor type 22 (lymphoid)), MYH14(myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin(protein C) 2, soluble (opsonic defect)), SELPLG (selectin P ligand),AOC3 (amine oxidase, copper containing 3 (vascular adhesion protein 1)),CTSL1 (cathepsin L1), PCNA (proliferating cell nuclear antigen), IGF2(insulin-like growth factor 2 (somatomedin A)), ITGB1 (integrin, beta 1(fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2,MSK12)), CAST (calpastatin), CXCL12 (chemokine (C-X-C motif) ligand 12(stromal cell-derived factor 1)), IGHE (immunoglobulin heavy constantepsilon), KCNE1 (potassium voltage-gated channel, Isk-related family,member 1), TFRC (transferrin receptor (p90, CD71)), COLlAl (collagen,type I, alpha 1), COL1A2 (collagen, type I, alpha 2), IL2RB (interleukin2 receptor, beta), PLA2G10 (phospholipase A2, group X), ANGPT2(angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4(NADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTPN11(protein tyrosine phosphatase, non-receptor type 11), SLC2A1 (solutecarrier family 2 (facilitated glucose transporter), member 1), IL2RA(interleukin 2 receptor, alpha), CCL5 (chemokine (C-C motif) ligand 5),IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-likeapoptosis regulator), CALCA (calcitonin-related polypeptide alpha),EIF4E (eukaryotic translation initiation factor 4E), GSTP1 (glutathioneS-transferase pi 1), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450,family 3, subfamily A, polypeptide 5), HSPG2 (heparan sulfateproteoglycan 2), CCL3 (chemokine (C-C motif) ligand 3), MYD88 (myeloiddifferentiation primary response gene (88)), VIP (vasoactive intestinalpeptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenergic, beta,receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member2), MMP8 (matrix metallopeptidase 8 (neutrophil collagenase)), NPR2(natriuretic peptide receptor B/guanylate cyclase B (atrionatriureticpeptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS(glutamyl-prolyl-tRNA synthetase), PPARGC1A (peroxisomeproliferator-activated receptor gamma, coactivator 1 alpha), F12(coagulation factor XII (Hageman factor)), PECAM1 (platelet/endothelialcell adhesion molecule), CCL4 (chemokine (C-C motif) ligand 4), SERPINA3(serpin peptidase inhibitor, clade A (alpha-1 antiproteinase,antitrypsin), member 3), CASR (calcium-sensing receptor), GJA5 (gapjunction protein, alpha 5, 40 kDa), FABP2 (fatty acid binding protein 2,intestinal), TTF2 (transcription termination factor, RNA polymerase II),PROS1 (protein S (alpha)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan,beta (43 kDa dystrophin-associated glycoprotein)), YME1L1 (YME1-like 1(S. cerevisiae)), CAMP (cathelicidin antimicrobial peptide), ZC3H12A(zinc finger CCCH-type containing 12A), AKR1B1 (aldo-keto reductasefamily 1, member B1 (aldose reductase)), DES (desmin), MMP7 (matrixmetallopeptidase 7 (matrilysin, uterine)), AHR (aryl hydrocarbonreceptor), CSF1 (colony stimulating factor 1 (macrophage)), HDAC9(histone deacetylase 9), CTGF (connective tissue growth factor), KCNMA1(potassium large conductance calcium-activated channel, subfamily M,alpha member 1), UGTlA (UDP glucuronosyltransferase 1 family,polypeptide A complex locus), PRKCA (protein kinase C, alpha), COMT(catechol-.beta.-methyltransferase), S100B (S100 calcium binding proteinB), EGR1 (early growth response 1), PRL (prolactin), IL15 (interleukin15), DRD4 (dopamine receptor D4), CAMK2G (calcium/calmodulin-dependentprotein kinase II gamma), SLC22A2 (solute carrier family 22 (organiccation transporter), member 2), CCL11 (chemokine (C-C motif) ligand 11),PGF (B321 placental growth factor), THPO (thrombopoietin), GP6(glycoprotein VI (platelet)), TACR1 (tachykinin receptor 1), NTS(neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1(potassium voltage-gated channel, Shal-related subfamily, member 1),LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1(platelet)), CYP2J2 (cytochrome P450, family 2, subfamily J, polypeptide2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol dehydrogenase 1C(class I), gamma polypeptide), ALOX12 (arachidonate 12-lipoxygenase),AHSG (alpha-2-HS-glycoprotein), BHMT (betaine-homocysteinemethyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa),SLC25A4 (solute carrier family 25 (mitochondrial carrier; adeninenucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP(arachidonate 5-lipoxygenase-activating protein), NUMA1 (nuclear mitoticapparatus protein 1), CYP27B1 (cytochrome P450, family 27, subfamily B,polypeptide 1), CYSLTR2 (cysteinyl leukotriene receptor 2), SOD3(superoxide dismutase 3, extracellular), LTC4S (leukotriene C4synthase), UCN (urocortin), GHRL (ghrelin/obestatin prepropeptide),APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4,member A), KBTBD10 (kelch repeat and BTB (POZ) domain containing 10),TNC (tenascin C), TYMS (thymidylate synthetase), SHCl (SHC (Src homology2 domain containing) transforming protein 1), LRP1 (low densitylipoprotein receptor-related protein 1), SOCS3 (suppressor of cytokinesignaling 3), ADH1B (alcohol dehydrogenase 1B (class I), betapolypeptide), KLK3 (kallikrein-related peptidase 3), HSDB11B1(hydroxysteroid (11-beta) dehydrogenase 1), VKORC1 (vitamin K epoxidereductase complex, subunit 1), SERPINB2 (serpin peptidase inhibitor,clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring fingerprotein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, alpha M(complement component 3 receptor 3 subunit)), PITX2 (paired-likehomeodomain 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (Fcfragment of IgG, low affinity 111a, receptor (CD16a)), LEPR (leptinreceptor), ENG (endoglin), GPX1 (glutathione peroxidase 1), GOT2(glutamic-oxaloacetic transaminase 2, mitochondrial (aspartateaminotransferase 2)), HRH1 (histamine receptor H1), NR112 (nuclearreceptor subfamily 1, group I, member 2), CRH (corticotropin releasinghormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1(voltage-dependent anion channel 1), HPSE (heparanase), SFTPD(surfactant protein D), TAP2 (transporter 2, ATP-binding cassette,sub-family B (MDR/TAP)), RNF123 (ring finger protein 123), PTK2B (PTK2Bprotein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase,receptor, type 2), IL6R (interleukin 6 receptor), ACHE(acetylcholinesterase (Yt blood group)), GLP1R (glucagon-like peptide 1receptor), GHR (growth hormone receptor), GSR (glutathione reductase),NQO1 (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptorsubfamily 5, group A, member 1), GJB2 (gap junction protein, beta 2, 26kDa), SLC9A1 (solute carrier family 9 (sodium/hydrogen exchanger),member 1), MAOA (monoamine oxidase A), PCSK9 (proprotein convertasesubtilisin/kexin type 9), FCGR2A (Fc fragment of IgG, low affinity Ha,receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, clade F (alpha-2antiplasmin, pigment epithelium derived factor), member 1), EDN3(endothelin 3), DHFR (dihydrofolate reductase), GAS6 (growtharrest-specific 6), SMPD1 (sphingomyelin phosphodiesterase 1, acidlysosomal), UCP2 (uncoupling protein 2 (mitochondrial, proton carrier)),TFAP2A (transcription factor AP-2 alpha (activating enhancer bindingprotein 2 alpha)), C4BPA (complement component 4 binding protein,alpha), SERPINF2 (serpin peptidase inhibitor, clade F (alpha-2antiplasmin, pigment epithelium derived factor), member 2), TYMP(thymidine phosphorylase), ALPP (alkaline phosphatase, placental (Reganisozyme)), CXCR2 (chemokine (C-X-C motif) receptor 2), SLC39A3 (solutecarrier family 39 (zinc transporter), member 3), ABCG2 (ATP-bindingcassette, sub-family G (WHITE), member 2), ADA (adenosine deaminase),JAK3 (Janus kinase 3), HSPA1A (heat shock 70 kDa protein 1A), FASN(fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), F11(coagulation factor XI), ATP7A (ATPase, Cu++ transporting, alphapolypeptide), CR1 (complement component (3b/4b) receptor 1 (Knops bloodgroup)), GFAP (glial fibrillary acidic protein), ROCK1 (Rho-associated,coiled-coil containing protein kinase 1), MECP2 (methyl CpG bindingprotein 2 (Rett syndrome)), MYLK (myosin light chain kinase), BCHE(butyrylcholinesterase), LIPE (lipase, hormone-sensitive), PRDX5(peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome.RecQ helicase-like), CXCR3 (chemokine (C-X-C motif) receptor 3), CD81(CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, gamma 2),MAP3K5 (mitogen-activated protein kinase kinase kinase 5), CHGA(chromogranin A (parathyroid secretory protein 1)), IAPP (islet amyloidpolypeptide), RHO (rhodopsin), ENPP1 (ectonucleotidepyrophosphatase/phosphodiesterase 1), PTHLH (parathyroid hormone-likehormone), NRG1 (neuregulin 1), VEGFC (vascular endothelial growth factorC), ENPEP (glutamyl aminopeptidase (aminopeptidase A)), CEBPB(CCAAT/enhancer binding protein (C/EBP), beta), NAGLU(N-acetylglucosaminidase, alpha-), F2RL3 (coagulation factor II(thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3-C motif) ligand 1),BDKRB1 (bradykinin receptor B1), ADAMTS13 (ADAM metallopeptidase withthrombospondin type 1 motif, 13), ELANE (elastase, neutrophilexpressed), ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2),CISH (cytokine inducible SH2-containing protein), GAST (gastrin), MYOC(myocilin, trabecular meshwork inducible glucocorticoid response),ATP1A2 (ATPase, Na+/K+ transporting, alpha 2 polypeptide), NF1(neurofibromin 1), GJB1 (gap junction protein, beta 1, 32 kDa), MEF2A(myocyte enhancer factor 2A), VCL (vinculin), BMPR2 (bone morphogeneticprotein receptor, type II (serine/threonine kinase)), TUBB (tubulin,beta), CDC42 (cell division cycle 42 (GTP binding protein, 25 kDa)),KRT18 (keratin 18), HSF1 (heat shock transcription factor 1), MYB (v-mybmyeloblastosis viral oncogene homolog (avian)), PRKAA2 (protein kinase,AMP-activated, alpha 2 catalytic subunit), ROCK2 (Rho-associated,coiled-coil containing protein kinase 2), TFPI (tissue factor pathwayinhibitor (lipoprotein-associated coagulation inhibitor)), PRKG1(protein kinase, cGMP-dependent, type I), BMP2 (bone morphogeneticprotein 2), CTNND1 (catenin (cadherin-associated protein), delta 1), CTH(cystathionase (cystathionine gamma-lyase)), CTSS (cathepsin S), VAV2(vav 2 guanine nucleotide exchange factor), NPY2R (neuropeptide Yreceptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36kDa), CD28 (CD28 molecule), GSTA1 (glutathione S-transferase alpha 1),PPIA (peptidylprolyl isomerase A (cyclophilin A)), APOH (apolipoproteinH (beta-2-glycoprotein I)), S100A8 (S100 calcium binding protein A8),IL11 (interleukin 11), ALOX15 (arachidonate 15-lipoxygenase), FBLN1(fibulin 1), NR1H3 (nuclear receptor subfamily 1, group H, member 3),SCD (stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastricinhibitory polypeptide), CHGB (chromogranin B (secretogranin 1)), PRKCB(protein kinase C, beta), SRD5A1 (steroid-5-alpha-reductase, alphapolypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1)),HSD11B2 (hydroxysteroid (11-beta) dehydrogenase 2), CALCRL (calcitoninreceptor-like), GALNT2 (UDP-N-acetyl-alpha-D-galactosamine:polypeptideN-acetylgalactosaminyltransferase 2 (GalNAc-T2)), ANGPTL4(angiopoietin-like 4), KCNN4 (potassium intermediate/small conductancecalcium-activated channel, subfamily N, member 4), PIK3C2A(phosphoinositide-3-kinase, class 2, alpha polypeptide), HBEGF(heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450,family 7, subfamily A, polypeptide 1), HLA-DRB5 (majorhistocompatibility complex, class II, DR beta 5), BNIP3 (BCL2/adenovirusEIB 19 kDa interacting protein 3), GCKR (glucokinase (hexokinase 4)regulator), S100A12 (S100 calcium binding protein A12), PADI4 (peptidylarginine deiminase, type IV), HSPA14 (heat shock 70 kDa protein 14),CXCR1 (chemokine (C-X-C motif) receptor 1), H19 (H19, imprintedmaternally expressed transcript (non-protein coding)), KRTAP19-3(keratin associated protein 19-3), IDDM2 (insulin-dependent diabetesmellitus 2), RAC2 (ras-related C3 botulinum toxin substrate 2 (rhofamily, small GTP binding protein Rac2)), RYR1 (ryanodine receptor 1(skeletal)), CLOCK (clock homolog (mouse)), NGFR (nerve growth factorreceptor (TNFR superfamily, member 16)), DBH (dopamine beta-hydroxylase(dopamine beta-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic,alpha 4), CACNA1C (calcium channel, voltage-dependent, L type, alpha 1Csubunit), PRKAG2 (protein kinase, AMP-activated, gamma 2 non-catalyticsubunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily 1, group H,member 2), TEK (TEK tyrosine kinase, endothelial), VEGFB (vascularendothelial growth factor B), MEF2C (myocyte enhancer factor 2C),MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2),TNFRSF11A (tumor necrosis factor receptor superfamily, member 11a, NFKBactivator), HSPA9 (heat shock 70 kDa protein 9 (mortalin)), CYSLTR1(cysteinyl leukotriene receptor 1), MAT1A (methionineadenosyltransferase I, alpha), OPRL1 (opiate receptor-like 1), IMPA1(inositol(myo)-1(or 4)-monophosphatase 1), CLCN2 (chloride channel 2),DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome (prosome,macropain) subunit, alpha type, 6), PSMB8 (proteasome (prosome,macropain) subunit, beta type, 8 (large multifunctional peptidase 7)),CHI3L1 (chitinase 3-like 1 (cartilage glycoprotein-39)), ALDH1B1(aldehyde dehydrogenase 1 family, member B1), PARP2 (poly (ADP-ribose)polymerase 2), STAR (steroidogenic acute regulatory protein), LBP(lipopolysaccharide binding protein), ABCC6 (ATP-binding cassette,sub-family C(CFTR/MRP), member 6), RGS2 (regulator of G-proteinsignaling 2, 24 kDa), EFNB2 (ephrin-B 2), GJB6 (gap junction protein,beta 6, 30 kDa), APOA2 (apolipoprotein A-II), AMPD1 (adenosinemonophosphate deaminase 1), DYSF (dysferlin, limb girdle musculardystrophy 2B (autosomal recessive)), FDFT1 (farnesyl-diphosphatefamesyltransferase 1), EDN2 (endothelin 2), CCR6 (chemokine (C-C motif)receptor 6), GJB3 (gap junction protein, beta 3, 31 kDa), IL1RL1(interleukin 1 receptor-like 1), ENTPD1 (ectonucleoside triphosphatediphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome 4), CELSR2 (cadherin,EGF LAG seven-pass G-type receptor 2 (flamingo homolog, Drosophila)),F11R (F11 receptor), RAPGEF3 (Rap guanine nucleotide exchange factor(GEF) 3), HYAL1 (hyaluronoglucosaminidase 1), ZNF259 (zinc fingerprotein 259), ATOX1 (ATX1 antioxidant protein 1 homolog (yeast)), ATF6(activating transcription factor 6), KHK (ketohexokinase(fructokinase)), SAT1 (spermidine/spermine N1-acetyltransferase 1), GGH(gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamylhydrolase)), TIMP4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solutecarrier family 4, sodium bicarbonate cotransporter, member 4), PDE2A(phosphodiesterase 2A, cGMP-stimulated), PDE3B (phosphodiesterase 3B,cGMP-inhibited), FADS1 (fatty acid desaturase 1), FADS2 (fatty aciddesaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxininteracting protein), LIMS1 (LIM and senescent cell antigen-like domains1), RHOB (ras homolog gene family, member B), LY96 (lymphocyte antigen96), FOXO1 (forkhead box O1), PNPLA2 (patatin-like phospholipase domaincontaining 2), TRH (thyrotropin-releasing hormone), GJC1 (gap junctionprotein, gamma 1, 45 kDa), SLC17A5 (solute carrier family 17(anion/sugar transporter), member 5), FTO (fat mass and obesityassociated), GJD2 (gap junction protein, delta 2, 36 kDa), PSRC1(proline/serine-rich coiled-coil 1), CASP12 (caspase 12(gene/pseudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK(PX domain containing serine/threonine kinase), IL33 (interleukin 33),TRIB1 (tribbles homolog 1 (Drosophila)), PBX4 (pre-B-cell leukemiahomeobox 4), NUPR1 (nuclear protein, transcriptional regulator, 1),15-September(15 kDa selenoprotein), CILP2 (cartilage intermediate layerprotein 2), TERC (telomerase RNA component), GGT2(gamma-glutamyltransferase 2), MT-COI (mitochondrially encodedcytochrome c oxidase I), and UOX (urate oxidase, pseudogene).

In an additional embodiment, the chromosomal sequence may further beselected from Pon1 (paraoxonase 1), LDLR (LDL receptor), ApoE(Apolipoprotein E), Apo B-100 (Apolipoprotein B-100), ApoA(Apolipoprotein(a)), ApoAl (Apolipoprotein A1), CBS (CystathioneB-synthase), Glycoprotein IIb/IIb, MTHRF (5,10-methylenetetrahydrofolatereductase (NADPH), and combinations thereof. In one iteration, thechromosomal sequences and proteins encoded by chromosomal sequencesinvolved in cardiovascular disease may be chosen from Cacna1C, Sod1,Pten, Ppar(alpha), Apo E, Leptin, and combinations thereof.

Lungs

The present invention also contemplates delivering the CRISPR-Cas systemto one or both lungs.

Although AAV-2-based vectors were originally proposed for CFTR deliveryto CF airways, other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9exhibit improved gene transfer efficiency in a variety of models of thelung epithelium (see, e.g., Li et al., Molecular Therapy, vol. 17 no.12, 2067-277 December 2009). AAV-1 was demonstrated to be ˜100-fold moreefficient than AAV-2 and AAV-5 at transducing human airway epithelialcells in vitro, 5 although AAV-1 transduced murine tracheal airwayepithelia in vivo with an efficiency equal to that of AAV-5. Otherstudies have shown that AAV-5 is 50-fold more efficient than AAV-2 atgene delivery to human airway epithelium (HAE) in vitro andsignificantly more efficient in the mouse lung airway epithelium invivo. AAV-6 has also been shown to be more efficient than AAV-2 in humanairway epithelial cells in vitro and murine airways in vivo. 8 The morerecent isolate, AAV-9, was shown to display greater gene transferefficiency than AAV-5 in murine nasal and alveolar epithelia in vivowith gene expression detected for over 9 months suggesting AAV mayenable long-term gene expression in vivo, a desirable property for aCFTR gene delivery vector. Furthermore, it was demonstrated that AAV-9could be readministered to the murine lung with no loss of CFTRexpression and minimal immune consequences. CF and non-CF HAE culturesmay be inoculated on the apical surface with 100 μl of AAV vectors forhours (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-277December 2009). The MOI may vary from 1×10³ to 4×10⁵ vectorgenomes/cell, depending on virus concentration and purposes of theexperiments. The above cited vectors are contemplated for the deliveryand/or administration of the invention.

Zamora et al. (Am J Respir Crit Care Med Vol 183. pp 531-538, 2011)reported an example of the application of an RNA interferencetherapeutic to the treatment of human infectious disease and also arandomized trial of an antiviral drug in respiratory syncytial virus(RSV)-infected lung transplant recipients. Zamora et al. performed arandomized, double-blind, placebocontrolled trial in LTX recipients withRSV respiratory tract infection. Patients were permitted to receivestandard of care for RSV. Aerosolized ALN-RSV01 (0.6 mg/kg) or placebowas administered daily for 3 days. This study demonstrates that an RNAitherapeutic targeting RSV can be safely administered to LTX recipientswith RSV infection. Three daily doses of ALN-RSV01 did not result in anyexacerbation of respiratory tract symptoms or impairment of lungfunction and did not exhibit any systemic proinflammatory effects, suchas induction of cytokines or CRP. Pharmacokinetics showed only low,transient systemic exposure after inhalation, consistent withpreclinical animal data showing that ALN-RSV01, administeredintravenously or by inhalation, is rapidly cleared from the circulationthrough exonucleasemediated digestion and renal excretion. The method ofZamora et al. may be applied to the CRISPR Cas system of the presentinvention and an aerosolized CRISPR Cas, for example with a dosage of0.6 mg/kg, may be contemplated for the present invention.

CFTRdelta508 chimeric guide RNA has been used for gene transfer or genedelivery of a CRISPR-Cas system in airways of subject or a patient inneed thereof, suffering from cystic fibrosis or from cystic fibrosis(CF) related symptoms, using adeno-associated virus (AAV) particles.This repair strategy exemplifies use for Cystic Fibrosis delta F508mutation. This type of strategy should apply across all organisms. Withparticular reference to CF, suitable patients may include: Human,non-primate human, canine, feline, bovine, equine and other domesticanimals. Applicants utilized a CRISPR-Cas system comprising a Cas9enzyme to target deltaF508 or other CFTR-inducing mutations.

The treated subjects in this instance receive pharmaceutically effectiveamount of aerosolized AAV vector system per lung endobronchiallydelivered while spontaneously breathing. As such, aerosolized deliveryis preferred for AAV delivery in general. An adenovirus or an AAVparticle may be used for delivery. Suitable gene constructs, eachoperably linked to one or more regulatory sequences, may be cloned intothe delivery vector. In this instance, the following constructs areprovided as examples: Cbh or EF1a promoter for Cas9, U6 or H1 promoterfor chimeric guide RNA): A preferred arrangement is to use aCFTRdelta508 targeting chimeric guide, a repair template for deltaF508mutation and a codon optimized Cas9 enzyme (preferred Cas9s are thosewith nuclease or nickase activity) with optionally one or more nuclearlocalization signal or sequence(s) (NLS(s)), e.g., two (2) NLSs.Constructs without NLS are also envisaged.

In order to identify the Cas9 target site, Applicants analyzed the humanCFTR genomic locus and identified the Cas9 target site. Preferably, ingeneral and in this CF case, the PAM may contain a NGG or a NNAGAAWmotif.

Accordingly, in the case of CF, the present method comprisesmanipulation of a target sequence in a genomic locus of interestcomprising delivering a non-naturally occurring or engineeredcomposition comprising a viral vector system comprising one or moreviral vectors operably encoding a composition for expression thereof,wherein the composition comprises:

-   -   a non-naturally occurring or engineered composition comprising a        vector system comprising one or more vectors comprising        -   I. a first regulatory element operably linked to a            CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide            sequence, wherein the polynucleotide sequence comprises            -   (a) a guide sequence capable of hybridizing to the CF                target sequence in a suitable mammalian cell,            -   (b) a tracr mate sequence, and            -   (c) a tracr sequence, and        -   II. a second regulatory element operably linked to an            enzyme-coding sequence encoding a CRISPR enzyme comprising            at least one or more nuclear localization sequences,            wherein (a), (b) and (c) are arranged in a 5′ to 3′            orientation,            wherein components I and II are located on the same or            different vectors of the system,            wherein when transcribed, the tracr mate sequence hybridizes            to the tracr sequence and the guide sequence directs            sequence-specific binding of a CRISPR complex to the target            sequence, and wherein the CRISPR complex comprises the            CRISPR enzyme complexed with (1) the guide sequence that is            hybridized to the target sequence, and (2) the tracr mate            sequence that is hybridized to the tracr sequence. In            respect of CF, preferred target DNA sequences comprise the            CFTRdelta508 mutation. A preferred PAM is described above. A            preferred CRISPR enzyme is any Cas. Alternatives to CF            include any genetic disorder and examples of these are well            known. Another preferred method or use of the invention is            for correcting defects in the EMP2A and EMP2B genes that            have been identified to be associated with Lafora disease.

In some embodiments, a “guide sequence” may be distinct from “guideRNA”. A guide sequence may refer to an approx. 20 bp sequence, withinthe guide RNA, that specifies the target site. In some embodiments, theCas9 is (or is derived from) SpCas9. In such embodiments, preferredmutations are at any or all or positions 10, 762, 840, 854, 863 and/or986 of SpCas9 or corresponding positions in other Cas9s (which may beascertained for instance by standard sequence comparison tools. Inparticular, any or all of the following mutations are preferred inSpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well asconservative substitution for any of the replacement amino acids is alsoenvisaged. The same (or conservative substitutions of these mutations)at corresponding positions in other Cas9s are also preferred.Particularly preferred are D10 and H840 in SpCas9. However, in otherCas9s, residues corresponding to SpCas9 D10 and H840 are also preferred.These are advantageous as they provide nickase activity. Such mutationsmay be applied to all aspects of the present invention, not onlytreatment of CF. Schwank et al. (Cell Stem Cell, 13:653-58, 2013) usedCRISPR/Cas9 to correct a defect associated with cystic fibrosis in humanstem cells. The team's target was the gene for an ion channel, cysticfibrosis transmembrane conductor receptor (CFTR). A deletion in CFTRcauses the protein to misfold in cystic fibrosis patients. Usingcultured intestinal stem cells developed from cell samples from twochildren with cystic fibrosis, Schwank et al. were able to correct thedefect using CRISPR along with a donor plasmid containing the reparativesequence to be inserted. The researchers then grew the cells intointestinal “organoids,” or miniature guts, and showed that theyfunctioned normally. In this case, about half of clonal organoidsunderwent the proper genetic correction.

Muscles

The present invention also contemplates delivering the CRISPR-Cas systemto muscle(s).

Bortolanza et al. (Molecular Therapy vol. 19 no. 11, 2055-264 November2011) shows that systemic delivery of RNA interference expressioncassettes in the FRG1 mouse, after the onset of facioscapulohumeralmuscular dystrophy (FSHD), led to a dose-dependent long-term FRG1knockdown without signs of toxicity. Bortolanza et al. found that asingle intravenous injection of 5×10¹² vg of rAAV6-sh1FRG1 rescuesmuscle histopathology and muscle function of FRG1 mice. In detail, 200μl containing 2×10¹² or 5×10¹² vg of vector in physiological solutionwere injected into the tail vein using a 25-gauge Terumo syringe. Themethod of Bortolanza et al. may be applied to an AAV expressing CRISPRCas and injected into humans at a dosage of about 2×10¹⁵ or 2×10¹⁶ vg ofvector.

Dumonceaux et al. (Molecular Therapy vol. 18 no. 5, 881-887 May 2010)inhibit the myostatin pathway using the technique of RNA interferencedirected against the myostatin receptor AcvRIIb mRNA (sh-AcvRIIb). Therestoration of a quasi-dystrophin was mediated by the vectorized U7exon-skipping technique (U7-DYS). Adeno-associated vectors carryingeither the sh-AcvrIIb construct alone, the U7-DYS construct alone, or acombination of both constructs were injected in the tibialis anterior(TA) muscle of dystrophic mdx mice. The injections were performed with10¹¹ AAV viral genomes. The method of Dumonceaux et al. may be appliedto an AAV expressing CRISPR Cas and injected into humans, for example,at a dosage of about 10¹⁴ to about 10¹⁵ vg of vector.

Kinouchi et al. (Gene Therapy (2008) 15, 1126-1130) report theeffectiveness of in vivo siRNA delivery into skeletal muscles of normalor diseased mice through nanoparticle formation of chemically unmodifiedsiRNAs with atelocollagen (ATCOL). ATCOL-mediated local application ofsiRNA targeting myostatin, a negative regulator of skeletal musclegrowth, in mouse skeletal muscles or intravenously, caused a markedincrease in the muscle mass within a few weeks after application. Theseresults imply that ATCOL-mediated application of siRNAs is a powerfultool for future therapeutic use for diseases including muscular atrophy.Mst-siRNAs (final concentration, 10 mM) were mixed with ATCOL (finalconcentration for local administration, 0.5%) (AteloGene, Kohken, Tokyo,Japan) according to the manufacturer's instructions. After anesthesia ofmice (20-week-old male C57BL/6) by Nembutal (25 mg/kg, i.p.), theMst-siRNA/ATCOL complex was injected into the masseter and bicepsfemoris muscles. The method of Kinouchi et al. may be applied to CRISPRCas and injected into a human, for example, at a dosage of about 500 to1000 ml of a 40 μM solution into the muscle.

Hagstrom et al. (Molecular Therapy Vol. 10, No. 2, August 2004) describean intravascular, nonviral methodology that enables efficient andrepeatable delivery of nucleic acids to muscle cells (myofibers)throughout the limb muscles of mammals. The procedure involves theinjection of naked plasmid DNA or siRNA into a distal vein of a limbthat is transiently isolated by a tourniquet or blood pressure cuff.Nucleic acid delivery to myofibers is facilitated by its rapid injectionin sufficient volume to enable extravasation of the nucleic acidsolution into muscle tissue. High levels of transgene expression inskeletal muscle were achieved in both small and large animals withminimal toxicity. Evidence of siRNA delivery to limb muscle was alsoobtained. For plasmid DNA intravenous injection into a rhesus monkey, athreeway stopcock was connected to two syringe pumps (Model PHD 2000;Harvard Instruments), each loaded with a single syringe. Five minutesafter a papaverine injection, pDNA (15.5 to 25.7 mg in 40-100 ml saline)was injected at a rate of 1.7 or 2.0 ml/s. This could be scaled up forplasmid DNA expressing CRISPR Cas of the present invention with aninjection of about 300 to 500 mg in 800 to 2000 ml saline for a human.For adenoviral vector injections into a rat, 2×10⁹ infectious particleswere injected in 3 ml of normal saline solution (NSS). This could bescaled up for an adenoviral vector expressing CRISPR Cas of the presentinvention with an injection of about 1×10¹³ infectious particles wereinjected in 10 liters of NSS for a human. For siRNA, a rat was injectedinto the great saphenous vein with 12.5 μg of a siRNA and a primate wasinjected injected into the great saphenous vein with 750 μg of a siRNA.This could be scaled up for a CRISPR Cas of the present invention, forexample, with an injection of about 15 to about 50 mg into the greatsaphenous vein of a human.

Skin

The present invention also contemplates delivering the CRISPR-Cas systemto the skin. Hickerson et al. (Molecular Therapy—Nucleic Acids (2013) 2,e129) relates to a motorized microneedle array skin delivery device fordelivering self-delivery (sd)-siRNA to human and murine skin. Theprimary challenge to translating siRNA-based skin therapeutics to theclinic is the development of effective delivery systems. Substantialeffort has been invested in a variety of skin delivery technologies withlimited success. In a clinical study in which skin was treated withsiRNA, the exquisite pain associated with the hypodermic needleinjection precluded enrollment of additional patients in the trial,highlighting the need for improved, more “patient-friendly” (i.e.,little or no pain) delivery approaches. Microneedles represent anefficient way to deliver large charged cargos including siRNAs acrossthe primary barrier, the stratum corneum, and are generally regarded asless painful than conventional hypodermic needles. Motorized “stamptype” microneedle devices, including the motorized microneedle array(MMNA) device used by Hickerson et al., have been shown to be safe inhairless mice studies and cause little or no pain as evidenced by (i)widespread use in the cosmetic industry and (ii) limited testing inwhich nearly all volunteers found use of the device to be much lesspainful than a flushot, suggesting siRNA delivery using this device willresult in much less pain than was experienced in the previous clinicaltrial using hypodermic needle injections. The MMNA device (marketed asTriple-M or Tri-M by Bomtech Electronic Co, Seoul, South Korea) wasadapted for delivery of siRNA to mouse and human skin. sd-siRNA solution(up to 300 μl of 0.1 mg/ml RNA) was introduced into the chamber of thedisposable Tri-M needle cartridge (Bomtech), which was set to a depth of0.1 mm. For treating human skin, deidentified skin (obtained immediatelyfollowing surgical procedures) was manually stretched and pinned to acork platform before treatment. All intradermal injections wereperformed using an insulin syringe with a 28-gauge 0.5-inch needle. TheMMNA device and method of Hickerson et al. could be used and/or adaptedto deliver the CRISPR Cas of the present invention, for example, at adosage of up to 300 μl of 0.1 mg/ml CRISPR Cas to the skin.

Leachman et al. (Molecular Therapy, vol. 18 no. 2, 442-446 February2010) relates to a phase Ib clinical trial for treatment of a rare skindisorder pachyonychia congenita (PC), an autosomal dominant syndromethat includes a disabling plantar keratoderma, utilizing the firstshort-interfering RNA (siRNA)-based therapeutic for skin. This siRNA,called TD101, specifically and potently targets the keratin 6a (K6a)N171K mutant mRNA without affecting wild-type K6a mRNA. Thedose-escalation schedule is presented below:

Concentration Total dose Volume of TD101 TD101 Week Dose no. Days (ml)(mg/ml) (mg) 1 1-2 1-7 0.1 1.0 0.10 2 3-4  8-14 0.25 1.0 0.25 3 5-615-21 0.50 1.0 0.50 4 7-8 22-28 1.0 1.0 1.0 5  9-10 29-35 1.5 1.0 1.5 611-12 36-42 2.0 1.0 2.0 7 13-14 43-49 2.0 1.5 3.0 8 15-16 50-56 2.0 2.04.0 9 17-18 57-63 2.0 2.5 5.0 10 19-20 64-70 2.0 3.0 6.0 11 21-22 71-772.0 3.5 7.0 12 23-24 78-84 2.0 4.0 8.0 13 25-26 85-91 2.0 4.5 9.0 1427-28 92-98 2.0 5.0 10.0 15 29-30  99-105 2.0 6.0 12.0 16 31-32 106-1122.0 7.0 14.0 17 33 113-119 2.0 8.5 17.0

Initially, 0.1 ml of a 1.0 mg/ml solution of TD101 or vehicle alone(Dulbecco's phosphate-buffered saline without calcium or magnesium) wasadministered to symmetric calluses. Six rising dose-volumes werecompleted without an adverse reaction to the increases: 0.1, 0.25, 0.5,1.0, 1.5, and 2.0 ml of a 1.0 mg/ml solution of TD101 solution perinjection. As the highest planned volume (2.0 ml) was well tolerated,the concentration of TD101 was then increased each week from 1 mg/ml upto a final concentration of 8.5 mg/ml. Similar dosages are contemplatedfor the administration of a CRISPR Cas that specifically and potentlytargets the keratin 6a (K6a) N171K mutant mRNA.

Zheng et al. (PNAS, Jul. 24, 2012, vol. 109, no. 30, 11975-11980) showthat spherical nucleic acid nanoparticle conjugates (SNA-NCs), goldcores surrounded by a dense shell of highly oriented, covalentlyimmobilized siRNA, freely penetrate almost 100% of keratinocytes invitro, mouse skin, and human epidermis within hours after application.Zheng et al. demonstrated that a single application of 25 nM epidermalgrowth factor receptor (EGFR) SNA-NCs for 60 h demonstrate effectivegene knockdown in human skin. A similar dosage may be contemplated forCRISPR Cas immobilized in SNA-NCs for administration to the skin.

Nucleic Acids, Amino Acids and Proteins, Regulatory Sequences, Vectors,Etc

Nucleic acids, amino acids and proteins: The invention uses nucleicacids to bind target DNA sequences. This is advantageous as nucleicacids are much easier and cheaper to produce than proteins, and thespecificity can be varied according to the length of the stretch wherehomology is sought. Complex 3-D positioning of multiple fingers, forexample is not required. The terms “polynucleotide”, “nucleotide”,“nucleotide sequence”, “nucleic acid” and “oligonucleotide” are usedinterchangeably. They refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three dimensional structure, andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: coding or non-coding regionsof a gene or gene fragment, loci (locus) defined from linkage analysis,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line. As used herein the term“variant” should be taken to mean the exhibition of qualities that havea pattern that deviates from what occurs in nature. The terms“non-naturally occurring” or “engineered” are used interchangeably andindicate the involvement of the hand of man. The terms, when referringto nucleic acid molecules or polypeptides mean that the nucleic acidmolecule or the polypeptide is at least substantially free from at leastone other component with which they are naturally associated in natureand as found in nature. “Complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick base pairing or other non-traditionaltypes. A percent complementarity indicates the percentage of residues ina nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.“Substantially complementary” as used herein refers to a degree ofcomplementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions. As used herein, “stringent conditions” forhybridization refer to conditions under which a nucleic acid havingcomplementarity to a target sequence predominantly hybridizes with thetarget sequence, and substantially does not hybridize to non-targetsequences. Stringent conditions are generally sequence-dependent, andvary depending on a number of factors. In general, the longer thesequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringentconditions are described in detail in Tijssen (1993), LaboratoryTechniques In Biochemistry And Molecular Biology-Hybridization WithNucleic Acid Probes Part I, Second Chapter “Overview of principles ofhybridization and the strategy of nucleic acid probe assay”, Elsevier,N.Y. Where reference is made to a polynucleotide sequence, thencomplementary or partially complementary sequences are also envisaged.These are preferably capable of hybridising to the reference sequenceunder highly stringent conditions. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 25° C. lower than the thermal melting point(T_(m)). The T_(m) is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 15° C. lowerthan the T_(m). In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 30° C. lower than the T_(m).Highly permissive (very low stringency) washing conditions may be as lowas 50° C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife—eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics. As used herein, the term “domain” or“protein domain” refers to a part of a protein sequence that may existand function independently of the rest of the protein chain. Asdescribed in aspects of the invention, sequence identity is related tosequence homology. Homology comparisons may be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences. In some preferred embodiments, the capping region of thedTALEs described herein have sequences that are at least 95% identicalor share identity to the capping region amino acid sequences providedherein. Sequence homologies may be generated by any of a number ofcomputer programs known in the art, for example BLAST or FASTA, etc. Asuitable computer program for carrying out such an alignment is the GCGWisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux etal., 1984, Nucleic Acids Research 12:387). Examples of other softwarethan may perform sequence comparisons include, but are not limited to,the BLAST package (see Ausubel et al., 1999 ibid—Chapter 18), FASTA(Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suiteof comparison tools. Both BLAST and FASTA are available for offline andonline searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60).However it is preferred to use the GCG Bestfit program. Percentage (%)sequence homology may be calculated over contiguous sequences, i.e., onesequence is aligned with the other sequence and each amino acid ornucleotide in one sequence is directly compared with the correspondingamino acid or nucleotide in the other sequence, one residue at a time.This is called an “ungapped” alignment. Typically, such ungappedalignments are performed only over a relatively short number ofresidues. Although this is a very simple and consistent method, it failsto take into consideration that, for example, in an otherwise identicalpair of sequences, one insertion or deletion may cause the followingamino acid residues to be put out of alignment, thus potentiallyresulting in a large reduction in % homology when a global alignment isperformed. Consequently, most sequence comparison methods are designedto produce optimal alignments that take into consideration possibleinsertions and deletions without unduly penalizing the overall homologyor identity score. This is achieved by inserting “gaps” in the sequencealignment to try to maximize local homology or identity. However, thesemore complex methods assign “gap penalties” to each gap that occurs inthe alignment so that, for the same number of identical amino acids, asequence alignment with as few gaps as possible—reflecting higherrelatedness between the two compared sequences—may achieve a higherscore than one with many gaps. “Affinity gap costs” are typically usedthat charge a relatively high cost for the existence of a gap and asmaller penalty for each subsequent residue in the gap. This is the mostcommonly used gap scoring system. High gap penalties may, of course,produce optimized alignments with fewer gaps. Most alignment programsallow the gap penalties to be modified. However, it is preferred to usethe default values when using such software for sequence comparisons.For example, when using the GCG Wisconsin Bestfit package the defaultgap penalty for amino acid sequences is −12 for a gap and −4 for eachextension. Calculation of maximum % homology therefore first requiresthe production of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4^(th) Ed. —Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol.403-410) and the GENEWORKS suite of comparison tools. Both BLAST andFASTA are available for offline and online searching (see Ausubel etal., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and thewebsite of the National Center for Biotechnology information at thewebsite of the National Institutes for Health). Although the final %homology may be measured in terms of identity, the alignment processitself is typically not based on an all-or-nothing pair comparison.Instead, a scaled similarity score matrix is generally used that assignsscores to each pair-wise comparison based on chemical similarity orevolutionary distance. An example of such a matrix commonly used is theBLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCGWisconsin programs generally use either the public default values or acustom symbol comparison table, if supplied (see user manual for furtherdetails). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62. Alternatively, percentagehomologies may be calculated using the multiple alignment feature inDNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL(Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the softwarehas produced an optimal alignment, it is possible to calculate %homology, preferably % sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult. The sequences may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in amino acidproperties (such as polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues) and it istherefore useful to group amino acids together in functional groups.Amino acids may be grouped together based on the properties of theirside chains alone. However, it is more useful to include mutation dataas well. The sets of amino acids thus derived are likely to be conservedfor structural reasons. These sets may be described in the form of aVenn diagram (Livingstone C. D. and Barton G. J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions may be made, for example accordingto the table below which describes a generally accepted Venn diagramgrouping of amino acids.

Set Sub-set Hydrophobic FWYHKMILVAGC Aromatic F W Y H Aliphatic I L VPolar WYHKREDCSTNQ Charged H K R E D Positively charged H K RNegatively charged E D Small VCAGSPTND Tiny A G S

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyriylalanine,thienylalanine, naphthylalanine and phenylglycine. Variant amino acidsequences may include suitable spacer groups that may be insertedbetween any two amino acid residues of the sequence including alkylgroups such as methyl, ethyl or propyl groups in addition to amino acidspacers such as glycine or β-alanine residues. A further form ofvariation, which involves the presence of one or more amino acidresidues in peptoid form, may be well understood by those skilled in theart. For the avoidance of doubt, “the peptoid form” is used to refer tovariant amino acid residues wherein the α-carbon substituent group is onthe residue's nitrogen atom rather than the α-carbon. Processes forpreparing peptides in the peptoid form are known in the art, for exampleSimon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, TrendsBiotechnol. (1995) 13(4), 132-134.

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR.

In certain aspects the invention involves vectors. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entityfrom one environment to another. It is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. Generally, avector is capable of replication when associated with the proper controlelements. In general, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Vectors include, but are not limited to, nucleic acidmolecules that are single-stranded, double-stranded, or partiallydouble-stranded; nucleic acid molecules that comprise one or more freeends, no free ends (e.g. circular); nucleic acid molecules that compriseDNA, RNA, or both; and other varieties of polynucleotides known in theart. One type of vector is a “plasmid,” which refers to a circulardouble stranded DNA loop into which additional DNA segments can beinserted, such as by standard molecular cloning techniques. Another typeof vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g. bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

Aspects of the invention relate to bicistronic vectors for chimeric RNAand Cas9. Bicistronic expression vectors for chimeric RNA and Cas9 arepreferred. In general and particularly in this embodiment Cas9 ispreferably driven by the CBh promoter. The chimeric RNA may preferablybe driven by a Pol III promoter, such as a U6 promoter. Ideally the twoare combined. The chimeric guide RNA typically consists of a 20 bp guidesequence (Ns) and this may be joined to the tracr sequence (running fromthe first “U” of the lower strand to the end of the transcript). Thetracr sequence may be truncated at various positions as indicated. Theguide and tracr sequences are separated by the tracr-mate sequence,which may be GUUUUAGAGCUA (SEQ ID NO: 42). This may be followed by theloop sequence GAAA as shown. Both of these are preferred examples.Applicants have demonstrated Cas9-mediated indels at the human EMX1 andPVALB loci by SURVEYOR assays. ChiRNAs are indicated by their “+n”designation, and crRNA refers to a hybrid RNA where guide and tracrsequences are expressed as separate transcripts. Throughout thisapplication, chimeric RNA may also be called single guide, or syntheticguide RNA (sgRNA). The loop is preferably GAAA, but it is not limited tothis sequence or indeed to being only 4 bp in length. Indeed, preferredloop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG. Inpracticing any of the methods disclosed herein, a suitable vector can beintroduced to a cell or an embryo via one or more methods known in theart, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g. transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g. liver,pancreas), or particular cell types (e.g. lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g. 1, 2,3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g.1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters(e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.Examples of pol III promoters include, but are not limited to, U6 and H1promoters. Examples of pol II promoters include, but are not limited to,the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally withthe RSV enhancer), the cytomegalovirus (CMV) promoter (optionally withthe CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],the SV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter. Also encompassed by the term “regulatory element” are enhancerelements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR ofHTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer;and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc.Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression desired, etc. A vectorcan be introduced into host cells to thereby produce transcripts,proteins, or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., clustered regularlyinterspersed short palindromic repeats (CRISPR) transcripts, proteins,enzymes, mutant forms thereof, fusion proteins thereof, etc.). Withregards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g. amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amrann etal., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 60-89). In some embodiments, a vector is a yeastexpression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943),pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif.). In some embodiments, a vector drives protein expression ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety. In some embodiments, a regulatoryelement is operably linked to one or more elements of a CRISPR system soas to drive expression of the one or more elements of the CRISPR system.In general, CRISPRs (Clustered Regularly Interspaced Short PalindromicRepeats), also known as SPIDRs (SPacer Interspersed Direct Repeats),constitute a family of DNA loci that are usually specific to aparticular bacterial species. The CRISPR locus comprises a distinctclass of interspersed short sequence repeats (SSRs) that were recognizedin E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; andNakata et al., J. Bacteriol., 171:3553-3556 [1989]), and associatedgenes. Similar interspersed SSRs have been identified in Haloferaxmediterranei, Streptococcus pyogenes, Anabaena, and Mycobacteriumtuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065 [1993];Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999]; Masepohl et al.,Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica et al., Mol.Microbiol., 17:85-93 [1995]). The CRISPR loci typically differ fromother SSRs by the structure of the repeats, which have been termed shortregularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol.,6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]).In general, the repeats are short elements that occur in clusters thatare regularly spaced by unique intervening sequences with asubstantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In some embodiments, the CRISPR enzyme is part of a fusion proteincomprising one or more heterologous protein domains (e.g. about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition tothe CRISPR enzyme). A CRISPR enzyme fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains. Examples of protein domains that may be fused to aCRISPR enzyme include, without limitation, epitope tags, reporter genesequences, and protein domains having one or more of the followingactivities: methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity and nucleic acid binding activity. Non-limiting examples ofepitope tags include histidine (His) tags, V5 tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx)tags. Examples of reporter genes include, but are not limited to,glutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP). ACRISPR enzyme may be fused to a gene sequence encoding a protein or afragment of a protein that bind DNA molecules or bind other cellularmolecules, including but not limited to maltose binding protein (MBP),S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domainfusions, and herpes simplex virus (HSV) BP16 protein fusions. Additionaldomains that may form part of a fusion protein comprising a CRISPRenzyme are described in US20110059502, incorporated herein by reference.In some embodiments, a tagged CRISPR enzyme is used to identify thelocation of a target sequence.

In some embodiments, a CRISPR enzyme may form a component of aninducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283, which is herebyincorporated by reference in its entirety.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Recombination Template (e.g., HDR Template)

In some embodiments, a recombination template is also provided. Arecombination template may be a component of another vector as describedherein, contained in a separate vector, or provided as a separatepolynucleotide. In some embodiments, a recombination template isdesigned to serve as a template in homologous recombination, such aswithin or near a target sequence nicked or cleaved by a CRISPR enzyme asa part of a CRISPR complex. A template polynucleotide may be of anysuitable length, such as about or more than about 10, 15, 20, 25, 50,75, 100, 150, 200, 500, 1000, or more nucleotides in length. In someembodiments, the template polynucleotide is complementary to a portionof a polynucleotide comprising the target sequence. When optimallyaligned, a template polynucleotide might overlap with one or morenucleotides of a target sequences (e.g. about or more than about 1, 5,10, 15, 20, or more nucleotides). In some embodiments, when a templatesequence and a polynucleotide comprising a target sequence are optimallyaligned, the nearest nucleotide of the template polynucleotide is withinabout 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000,10000, or more nucleotides from the target sequence.

Modifying a Target

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal, or a plant, andmodifying the cell or cells. Culturing may occur at any stage ex vivo.The cell or cells may even be re-introduced into the non-human animal orplant. For re-introduced cells it is particularly preferred that thecells are stem cells.

In some embodiments, the method comprises allowing a CRISPR complex tobind to the target polynucleotide to effect cleavage of said targetpolynucleotide thereby modifying the target polynucleotide, wherein theCRISPR complex comprises a CRISPR enzyme complexed with a guide sequencehybridized to a target sequence within said target polynucleotide,wherein said guide sequence is linked to a tracr mate sequence which inturn hybridizes to a tracr sequence.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPRenzyme complexed with a guide sequence hybridized to a target sequencewithin said polynucleotide, wherein said guide sequence is linked to atracr mate sequence which in turn hybridizes to a tracr sequence.Similar considerations and conditions apply as above for methods ofmodifying a target polynucleotide. In fact, these sampling, culturingand re-introduction options apply across the aspects of the presentinvention.

Indeed, in any aspect of the invention, the CRISPR complex may comprisea CRISPR enzyme complexed with a guide sequence hybridized to a targetsequence, wherein said guide sequence may be linked to a tracr matesequence which in turn may hybridize to a tracr sequence. Similarconsiderations and conditions apply as above for methods of modifying atarget polynucleotide.

Kits

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. Elementsmay be provided individually or in combinations, and may be provided inany suitable container, such as a vial, a bottle, or a tube. In someembodiments, the kit includes instructions in one or more languages, forexample in more than one language.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein. Reagentsmay be provided in any suitable container. For example, a kit mayprovide one or more reaction or storage buffers. Reagents may beprovided in a form that is usable in a particular assay, or in a formthat requires addition of one or more other components before use (e.g.in concentrate or lyophilized form). A buffer can be any buffer,including but not limited to a sodium carbonate buffer, a sodiumbicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, aHEPES buffer, and combinations thereof. In some embodiments, the bufferis alkaline. In some embodiments, the buffer has a pH from about 7 toabout 10. In some embodiments, the kit comprises one or moreoligonucleotides corresponding to a guide sequence for insertion into avector so as to operably link the guide sequence and a regulatoryelement. In some embodiments, the kit comprises a homologousrecombination template polynucleotide. In some embodiments, the kitcomprises one or more of the vectors and/or one or more of thepolynucleotides described herein. The kit may advantageously allows toprovide all elements of the systems of the invention.

Disease-Associated Genes and Polynucleotides

Examples of disease-associated genes and polynucleotides that can betargeted in the practice of the invention are listed in Tables A and B.Disease specific information is available from McKusick-NathansInstitute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)and National Center for Biotechnology Information, National Library ofMedicine (Bethesda, Md.), available on the World Wide Web. Examples ofsignaling biochemical pathway-associated genes and polynucleotides arelisted in Table C. Mutations in these genes and pathways can result inproduction of improper proteins or proteins in improper amounts whichaffect function. Further examples of genes, diseases and proteins arehereby incorporated by reference from U.S. Provisional application61/736,527 filed Dec. 12, 2012. Such genes, proteins and pathways may bethe target polynucleotide of a CRISPR complex.

TABLE A DISEASE/DISORDERS GENE(S) Neoplasia PTEN; ATM; ATR; EGFR; ERBB2;ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF;HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (Wilms Tumor);FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB(retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (Androgen Receptor);TSG101; IGF; IGF Receptor; Igf1 (4 variants); Igf2 (3 variants); Igf 1Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2,3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related Macular Abcr; Ccl2; Cc2; cp(ceruloplasmin); Timp3; cathepsinD; Degeneration Vldlr; Ccr2Schizophrenia Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin);Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophanhydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b Disorders 5-HTT (Slc6a4);COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1) Trinucleotide HTT(Huntington's Dx); SBMA/SMAX1/AR (Kennedy's Repeat Disorders Dx);FXN/X25 (Friedrich's Ataxia); ATX3 (Machado- Joseph's Dx); ATXN1 andATXN2 (spinocerebellar ataxias); DMPK (myotonic dystrophy); Atrophin-1and Atn1 (DRPLA Dx); CBP (Creb-BP - global instability); VLDLR(Alzheimer's); Atxn7; Atxn10 Fragile X Syndrome FMR2; FXR1; FXR2; mGLUR5Secretase Related APH-1 (alpha and beta); Presenilin (Psen1); nicastrinDisorders (Ncstn); PEN-2 Others Nos1; Parp1; Nat1; Nat2 Prion - relateddisorders Prp ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b;VEGF-c) Drug addiction Prkce (alcohol); Drd2; Drd4; ABAT (alcohol);GRIA2; Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol) AutismMecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1;FXR2; Mglur5) Alzheimer's Disease E1; CHIP; UCH; UBB; Tau; LRP; PICALM;Clusterin; PS1; SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28 (Aqp1, Aquaporin1); Uchl1; Uchl3; APP Inflammation IL-10; IL-1 (IL-1a; IL-1b); IL-13;IL-17 (IL-17a (CTLA8); IL- 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1;ptpn22; TNFa; NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4;Cx3cl1 Parkinson's Disease x-Synuclein; DJ-1; LRRK2; Parkin; PINK1

TABLE B Blood and Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3,UMPH1, coagulation diseases PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2,ANH1, ASB, and disorders ABCB7, ABC7, ASAT); Bare lymphocyte syndrome(TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP,RFX5), Bleeding disorders (TBXA2R, P2RX1, P2X1); Factor H and factorH-like 1 (HF1, CFH, HUS); Factor V and factor VIII (MCFD2); Factor VIIdeficiency (F7); Factor X deficiency (F10); Factor XI deficiency (F11);Factor XII deficiency (F12, HAF); Factor XIIIA deficiency (F13A1, F13A);Factor XIIIB deficiency (F13B); Fanconi anemia (FANCA, FACA, FA1, FA,FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1,FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1,BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); Hemophagocyticlymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3,HLH3, FHL3); Hemophilia A (F8, F8C, HEMA); Hemophilia B (F9, HEMB),Hemorrhagic disorders (PI, ATT, F5); Leukocyde deficiencies anddisorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3,EIF2B5, LVWM, CACH, CLE, EIF2B4); Sickle cell anemia (HBB); Thalassemia(HBA2, HBB, HBD, LCRB, HBA1). Cell dysregulation B-cell non-Hodgkinlymphoma (BCL7A, BCL7); Leukemia (TAL1 and oncology TCL5, SCL, TAL2,FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, diseases and disorders HOXD4,HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12,LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT,LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3,FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM,CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF,WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA,GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN,CAIN). Inflammation and AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1,IFNG, CXCL12, immune related SDF1); Autoimmune lymphoproliferativesyndrome (TNFRSF6, APT1, diseases and disorders FAS, CD95, ALPS1A);Combined immunodeficiency, (IL2RG, SCIDX1, SCIDX, IMD4); HIV-1 (CCL5,SCYA5, D17S136E, TCP228), HIV susceptibility or infection (IL10, CSIF,CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G,AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG,HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI);Inflammation (IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8),IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa,NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1);Severe combined immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS,SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG,SCIDX1, SCIDX, IMD4). Metabolic, liver, Amyloid neuropathy (TTR, PALB);Amyloidosis (APOA1, APP, AAA, kidney and protein CVAP, AD1, GSN, FGA,LYZ, TTR, PALB); Cirrhosis (KRT18, KRT8, diseases and disorders CIRH1A,NAIC, TEX292, KIAA1988); Cystic fibrosis (CFTR, ABCC7, CF, MRP7);Glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2,LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); Hepatic adenoma, 142330 (TCF1,HNF1A, MODY3), Hepatic failure, early onset, and neurologic disorder(SCOD1, SCO1), Hepatic lipase deficiency (LIPC), Hepatoblastoma, cancerand carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53,P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; Medullary cystic kidneydisease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); Phenylketonuria (PAH, PKU1,QDPR, DHPR, PTS); Polycystic kidney and hepatic disease (FCYT, PKHD1,ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63).Muscular/Skeletal Becker muscular dystrophy (DMD, BMD, MYF6), DuchenneMuscular diseases and disorders Dystrophy (DMD, BMD); Emery-Dreifussmuscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA,LMN1, EMD2, FPLD, CMD1A); Facioscapulohumeral muscular dystrophy(FSHMD1A, FSHD1A); Muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM,LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B,SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E,SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H,FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C,SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); Osteopetrosis (LRP5, BMND1,LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7,OC116, OPTB1); Muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2,SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2,CATF1, SMARD1). Neurological and ALS (SOD1, ALS2, STEX, FUS, TARDBP,VEGF (VEGF-a, VEGF-b, neuronal diseases VEGF-c); Alzheimer disease (APP,AAA, CVAP, AD1, APOE, AD2, and disorders PSEN2, AD4, STM2, APBB2,FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP,A2M, BLMH, BMH, PSEN1, AD3); Autism (Mecp2, BZRAP1, MDGA2, Sema5A,Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4,KIAA1260, AUTSX2); Fragile X Syndrome (FMR2, FXR1, FXR2, mGLUR5);Huntington's disease and disease like disorders (HD, IT15, PRNP, PRIP,JPH3, JP3, HDL2, TBP, SCA17); Parkinson disease (NR4A2, NURR1, NOT,TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2,PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN,PARK2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79,CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1);Schizophrenia (Neuregulin1 (Nrg1), Erb4 (receptor for Neuregulin),Complexin1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophanhydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD(Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1)); Secretase Related Disorders(APH-1 (alpha and beta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2,Nos1, Parp1, Nat1, Nat2); Trinucleotide Repeat Disorders (HTT(Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich'sAtaxia), ATX3 (Machado- Joseph's Dx), ATXN1 and ATXN2 (spinocerebellarataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP(Creb-BP - global instability), VLDLR (Alzheimer's), Atxn7, Atxn10).Occular diseases Age-related macular degeneration (Abcr, Ccl2, Cc2, cp(ceruloplasmin), and disorders Timp3, cathepsinD, Vldlr, Ccr2); Cataract(CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1,PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD,CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2,CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA,CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1);Corneal clouding and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3,CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD,PPCD2, PIP5K3, CFD); Cornea plana congenital (KERA, CNA2); Glaucoma(MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1,GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A); Leber congenital amaurosis (CRB1,RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4,GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); Macular dystrophy (ELOVL4,ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

TABLE C CELLULAR FUNCTION GENES PI3K/AKT Signaling PRKCE; ITGAM; ITGA5;IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1;AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8;BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1;MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB;DYRK1A; CDKN1A; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1;PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN;ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KB1ERK/MAPK Signaling PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2;RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA;CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8;MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9;SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1;FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3;ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF;STAT1; SGK Glucocorticoid Receptor RAC1; TAF4B; EP300; SMAD2; TRAF6;PCAF; ELK1; Signaling MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA;CREB1; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8;BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A;MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3;MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8;NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1;SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 AxonalGuidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; Signaling IGF1;RAC1; RAP1A; E1F4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF;RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ;PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS;RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2;PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3;CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA EphrinReceptor PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; Signaling PRKAA2;EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1;AKT2; DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8;GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2;PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4, AKT1; JAK2;STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK;CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK Actin Cytoskeleton ACTN4;PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; Signaling PRKAA2; EIF2AK2; RAC1; INS;ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1;PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS;RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN;VIL2; RAF1; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1;PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGKHuntington's Disease PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2;Signaling MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5;CREB1; PRKC1; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1;GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11;MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1;CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK;HDAC6; CASP3 Apoptosis Signaling PRKCE; ROCK1; BID; IRAK1; PRKAA2;EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2;CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8;KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG;RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA;CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 BCell Receptor RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; SignalingAKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3;MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9;EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1;PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN;GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Leukocyte Extravasation ACTN4; CD44;PRKCE; ITGAM; ROCK1; CXCR4; CYBA; Signaling RAC1; RAP1A; PRKCZ; ROCK2;RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8;PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A;BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1;CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9Integrin Signaling ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1;ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3;MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7;PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1;TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3Acute Phase Response IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11;Signaling AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8;RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1;TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2;AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3;IL1R1; IL6 PTEN Signaling ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11;MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2;PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1;IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1;MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1;CASP3; RPS6KB1 p53 Signaling PTEN; EP300; BBC3; PCAF; FASN; BRCA1;GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3;MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1;HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1;RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2;GSK3B; BAX; AKT3 Aryl Hydrocarbon HSPB1; EP300; FASN; TGM2; RXRA; MAPK1;NQO1; Receptor NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; Signaling SMARCA4;NFKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73;GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2;APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6;CYP1B1; HSP90AA1 Xenobiotic Metabolism PRKCE; EP300; PRKCZ; RXRA; MAPK1;NQO1; Signaling NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB;PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13;PRKCD; GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A;PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1;NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK SignalingPRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2;PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1;IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1;PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3;CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK PPAr/RXR Signaling PRKAA2;EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB;NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS;RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7;CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1;PRKCA; IL6; HSP90AA1; ADIPOQ NF-KB Signaling IRAK1; EIF2AK2; EP300; INS;MYD88; PRKCZ: TRAF6; TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2;MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A;TRAF2; TLR4: PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1;PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1Neuregulin Signaling ERBB4; PRKCE; ITGAM; ITGA5: PTEN; PRKCZ; ELK1;MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3;ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17;AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC;NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt & Beta catenin CD44;EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; Signaling AKT2; PIN1; CDH1; BTRC;GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2: ILK; LEF1;SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1;TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2Insulin Receptor PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; SignalingPTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3;TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2;JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B;AKT3; FOXO1; SGK; RPS6KB1 IL-6 Signaling HSPB1; TRAF6; MAPKAPK2; ELK1;MAPK1; PTPN11; IKBKB; FOS; NFKB2: MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST;KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1;IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1;CEBPB; JUN; IL1R1; SRF; IL6 Hepatic Cholestasis PRKCE; IRAK1; INS;MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8;PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG;RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN;IL1R1; PRKCA; IL6 IGF-1 Signaling IGF1; PRKCZ; ELK1; MAPK1; PTPN11;NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R;IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2;AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF;CTGF; RPS6KB1 NRF2-mediated PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1;Oxidative NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8; StressResponse PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A;MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN;KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic Fibrosis/HepaticEDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; Stellate Cell ActivationSMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4;PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1;CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR Signaling EP300; INS;TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B;MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF;INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1;NFKB1; JUN; IL1R1; HSP90AA1 Fc Epsilon RI Signaling PRKCE; RAC1; PRKCZ;LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK;MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VAV3;PRKCA G-Protein Coupled PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB;Receptor Signaling PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3;MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1;PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCAInositol Phosphate PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; MetabolismMAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD;PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1;MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK PDGF Signaling EIF2AK2; ELK1;ABL2; MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3;KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA;STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGFSignaling ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA;ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3;PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA;AKT3; FOXO1; PRKCA Natural Killer Cell PRKCE; RAC1; PRKCZ; MAPK1; RAC2;PTPN11; Signaling KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3;PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4;AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell Cycle: G1/S HDAC4;SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; Checkpoint Regulation ATR; ABL1;E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53;CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1;HDAC6 T Cell Receptor RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS;Signaling NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA, PIK3C2A; BTK;LCK; RAF1; IKBKG; RELB, FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK;BCL10; JUN; VAV3 Death Receptor Signaling CRADD; HSPB1; BID; BIRC4;TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX;TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1;CASP2; BIRC2; CASP3; BIRC3 FGF Signaling RAC1; FGFR1; MET; MAPKAPK2;MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3;MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1;FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF GM-CSF Signaling LYN; ELK1; MAPK1;PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1;MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic Lateral BID; IGF1;RAC1; BIRC4; PGF; CAPNS1; CAPN2; Sclerosis Signaling PIK3CA; BCL2;PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A;CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat SignalingPTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS;SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2;PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1 Nicotinate and PRKCE; IRAK1;PRKAA2; EIF2AK2; GRK6; MAPK1; Nicotinamide PLK1; AKT2; CDK8; MAPK8;MAPK3; PRKCD; PRKAA1; Metabolism PBEF1; MAPK9; CDK2; PIM1; DYRK1A;MAP2K2; MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine SignalingCXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8;MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1;MAP2K2; MAP2K1; JUN; CCL2; PRKCA IL-2 Signaling ELK1; MAPK1; PTPN11;AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS;SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1;JUN; AKT3 Synaptic Long Term PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1;GNAS; Depression PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN;PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCAEstrogen Receptor TAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; SignalingSMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3;RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 ProteinUbiquitination TRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; Pathway CBL;UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2;PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3 IL-10 Signaling TRAF6; CCR1;ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF;IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6 VDR/RXRActivation PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKCI;CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1;LRP5; CEBPB; FOXO1; PRKCA TGF-beta Signaling EP300; SMAD2; SMURF1;MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1;RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5Toll-like Receptor IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; SignalingIKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG;RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN p38 MAPK Signaling HSPB1; IRAK1;TRAF6; MAPKAPK2; ELK1; FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13;TRAF2; MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1Neurotrophin/TRK NTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS; SignalingPIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1;PDPK1; MAP2K1; CDC42; JUN; ATF4 FXR/RXR Activation INS; PPARA; FASN;RXRA; AKT2; SDC1; MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A;TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1 Synaptic Long Term PRKCE;RAP1A; EP300; PRKCZ; MAPK1; CREB1; Potentiation PRKCI; GNAQ; CAMK2A;PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4;PRKCA Calcium Signaling RAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1;CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR;CAMKK2; ATF4; HDAC6 EGF Signaling ELK1; MAPK1; EGFR; PIK3CA; FOS;PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; STAT3;MAP2K1; JUN; PRKCA; SRF; STAT1 Hypoxia Signaling in the EDN1; PTEN;EP300; NQO1; UBE2I; CREB1; ARNT; Cardiovascular System HIF1A; SLC2A4;NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; HSP90AA1 LPS/IL-1Mediated IRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1, Inhibition MAPK8;ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2; of RXR Function TLR4; TNF; MAP3K7;NR1H2; SREBF1; JUN; IL1R1 LXR/RXR Activation FASN; RXRA; NCOR2; ABCA1;NFKB2; IRF3; RELA; NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1;IL1R1; CCL2; IL6; MMP9 Amyloid Processing PRKCE; CSNK1E; MAPK1; CAPNS1;AKT2; CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1;GSK3B; AKT3; APP IL-4 Signaling AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1;KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1;AKT3; RPS6KB1 Cell Cycle: G2/M DNA EP300; PCAF; BRCA1; GADD45A; PLK1;BTRC; Damage Checkpoint CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A;Regulation PRKDC; ATM; SFN; CDKN2A Nitric Oxide Signaling in KDR; FLT1;PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; the Cardiovascular System CAV1;PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; VEGFA; AKT3; HSP90AA1 PurineMetabolism NME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4; PKM2; ENTPD1;RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1 cAMP-mediated RAP1A;MAPK1; GNAS; CREB1; CAMK2A; MAPK3; Signaling SRC; RAF1; MAP2K2; STAT3;MAP2K1; BRAF; ATF4 Mitochondrial SOD2; MAPK8; CASP8; MAPK10; MAPK9;CASP9; Dysfunction PARK7; PSEN1; PARK2; APP; CASP3 Notch Signaling HES1;JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3; NOTCH1; DLL4Endoplasmic Reticulum HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4;Stress Pathway EIF2AK3; CASP3 Pyrimidine Metabolism NME2; AICDA; RRM2;EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson's Signaling UCHL1;MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3 Cardiac & Beta GNAS;GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; Adrenergic Signaling PPP2R5CGlycolysis/ HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1 GluconeogenesisInterferon Signaling IRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; IFIT3 SonicHedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B; DYRKIB SignalingGlycerophospholipid PLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2 MetabolismPhospholipid PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2 DegradationTryptophan Metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; SIAH1 LysineDegradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C Nucleotide ExcisionERCC5; ERCC4; XPA; XPC; ERCC1 Repair Pathway Starch and Sucrose UCHL1;HK2; GCK; GPI; HK1 Metabolism Aminosugars Metabolism NQO1; HK2; GCK; HK1Arachidonic Acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism Circadian RhythmCSNK1E; CREB1; ATF4; NR1D1 Signaling Coagulation System BDKRB1; F2R;SERPINE1; F3 Dopamine Receptor PPP2R1A; PPP2CA; PPP1CC; PPP2R5CSignaling Glutathione Metabolism IDH2; GSTP1; ANPEP; IDH1 GlycerolipidMetabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic Acid Metabolism PRDX6;GRN; YWHAZ; CYP1B1 Methionine Metabolism DNMT1; DNMT3B; AHCY; DNMT3APyruvate Metabolism GLO1; ALDH1A1; PKM2; LDHA Arginine and ProlineALDH1A1; NOS3; NOS2A Metabolism Eicosanoid Signaling PRDX6; GRN; YWHAZFructose and Mannose HK2; GCK; HK1 Metabolism Galactose Metabolism HK2;GCK; HK1 Stilbene, Coumarine and PRDX6; PRDX1; TYR Lignin BiosynthesisAntigen Presentation CALR; B2M Pathway Biosynthesis of Steroids NQO1;DHCR7 Butanoate Metabolism ALDH1A1; NLGN1 Citrate Cycle IDH2; IDH1 FattyAcid Metabolism ALDH1A1; CYP1B1 Glycerophospholipid PRDX6; CHKAMetabolism Histidine Metabolism PRMT5; ALDH1A1 Inositol MetabolismERO1L; APEX1 Metabolism of GSTP1; CYP1B1 Xenobiotics by Cytochrome p450Methane Metabolism PRDX6; PRDX1 Phenylalanine PRDX6; PRDX1 MetabolismPropanoate Metabolism ALDH1A1; LDHA Selenoamino Acid PRMT5; AHCYMetabolism Sphingolipid Metabolism SPHK1; SPHK2 Aminophosphonate PRMT5Metabolism Androgen and Estrogen PRMT5 Metabolism Ascorbate and AldarateALDH1A1 Metabolism Bile Acid Biosynthesis ALDH1A1 Cysteine MetabolismLDHA Fatty Acid Biosynthesis FASN Glutamate Receptor GNB2L1 SignalingNRF2-mediated PRDX1 Oxidative Stress Response Pentose Phosphate GPIPathway Pentose and Glucuronate UCHL1 Interconversions RetinolMetabolism ALDH1A1 Riboflavin Metabolism TYR Tyrosine Metabolism PRMT5,TYR Ubiquinone Biosynthesis PRMT5 Valine, Leucine and ALDH1A1 IsoleucineDegradation Glycine, Serine and CHKA Threonine Metabolism LysineDegradation ALDH1A1 Pain/Taste TRPM5; TRPA1 Pain TRPM7; TRPC5; TRPC6;TRPC1; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5;Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial Function AIF; CytC; SMAC(Diablo); Aifm-1; Aifm-2 Developmental BMP-4; Chordin (Chrd); Noggin(Nog); WNT (Wnt2; Neurology Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b;Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; Dkk-1;Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86(Pou4f1 or Brn3a); Numb; Reln

Embodiments of the invention also relate to methods and compositionsrelated to knocking out genes, amplifying genes and repairing particularmutations associated with DNA repeat instability and neurologicaldisorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities andNeurological Diseases, Second Edition, Academic Press, Oct. 13,2011—Medical). Specific aspects of tandem repeat sequences have beenfound to be responsible for more than twenty human diseases (Newinsights into repeat instability: role of RNA⋅DNA hybrids. McIvor E I,Polak U, Napierala M. RNA Biol. 2010 September-October; 7(5):551-8). TheCRISPR-Cas system may be harnessed to correct these defects of genomicinstability.

A further aspect of the invention relates to utilizing the CRISPR-Cassystem for correcting defects in the EMP2A and EMP2B genes that havebeen identified to be associated with Lafora disease. Lafora disease isan autosomal recessive condition which is characterized by progressivemyoclonus epilepsy which may start as epileptic seizures in adolescence.A few cases of the disease may be caused by mutations in genes yet to beidentified. The disease causes seizures, muscle spasms, difficultywalking, dementia, and eventually death. There is currently no therapythat has proven effective against disease progression. Other geneticabnormalities associated with epilepsy may also be targeted by theCRISPR-Cas system and the underlying genetics is further described inGenetics of Epilepsy and Genetic Epilepsies, edited by GiulianoAvanzini, Jeffrey L. Noebels, Mariani Foundation PaediatricNeurology:20; 2009).

The methods of US Patent Publication No. 20110158957 assigned to SangamoBioSciences, Inc. involved in inactivating T cell receptor (TCR) genesmay also be modified to the CRISPR Cas system of the present invention.In another example, the methods of US Patent Publication No. 20100311124assigned to Sangamo BioSciences, Inc. and US Patent Publication No.20110225664 assigned to Cellectis, which are both involved ininactivating glutamine synthetase gene expression genes may also bemodified to the CRISPR Cas system of the present invention.

Several further aspects of the invention relate to correcting defectsassociated with a wide range of genetic diseases which are furtherdescribed on the website of the National Institutes of Health under thetopic subsection Genetic Disorders (website athealth.nih.gov/topic/GeneticDisorders). The genetic brain diseases mayinclude but are not limited to Adrenoleukodystrophy, Agenesis of theCorpus Callosum, Aicardi Syndrome, Alpers' Disease, Alzheimer's Disease,Barth Syndrome, Batten Disease, CADASIL, Cerebellar Degeneration,Fabry's Disease, Gerstmann-Straussler-Scheinker Disease, Huntington'sDisease and other Triplet Repeat Disorders, Leigh's Disease, Lesch-NyhanSyndrome, Menkes Disease, Mitochondrial Myopathies and NINDSColpocephaly. These diseases are further described on the website of theNational Institutes of Health under the subsection Genetic BrainDisorders.

In Cartier, “MINI-SYMPOSIUM: X-Linked Adrenoleukodystrophypa,Hematopoietic Stem Cell Transplantation and Hematopoietic Stem Cell GeneTherapy in X-Linked Adrenoleukodystrophy,” Brain Pathology 20 (2010)857-862, incorporated herein by reference along with the documents itcites, as if set out in full, there is recognition that allogeneichematopoietic stem cell transplantation (HSCT) was utilized to delivernormal lysosomal enzyme to the brain of a patient with Hurler's disease,and a discussion of HSC gene therapy to treat ALD. In two patients,peripheral CD34+ cells were collected after granulocyte-colonystimulating factor (G-CSF) mobilization and transduced with anmyeloproliferative sarcoma virus enhancer, negative control regiondeleted, dl587rev primer binding site substituted (MND)-ALD lentiviralvector. CD34+ cells from the patients were transduced with the MND-ALDvector during 16 h in the presence of cytokines at low concentrations.Transduced CD34+ cells were frozen after transduction to perform on 5%of cells various safety tests that included in particular threereplication-competent lentivirus (RCL) assays. Transduction efficacy ofCD34+ cells ranged from 35% to 50% with a mean number of lentiviralintegrated copy between 0.65 and 0.70. After the thawing of transducedCD34+ cells, the patients were reinfused with more than 4.10⁶ transducedCD34+ cells/kg following full myeloablation with busulfan andcyclophos-phamide. The patient's HSCs were ablated to favor engraftmentof the gene-corrected HSCs. Hematological recovery occurred between days13 and 15 for the two patients. Nearly complete immunological recoveryoccurred at 12 months for the first patient, and at 9 months for thesecond patient. In contrast to using lentivirus, with the knowledge inthe art and the teachings in this disclosure, the skilled person cancorrect HSCs as to ALD using a CRISPR-Cas9 system that targets andcorrects the mutation (e.g., with a suitable HDR template);specifically, the sgRNA can target mutations in ABCD1, a gene located onthe X chromosome that codes for ALD, a peroxisomal membrane transporterprotein, and the HDR can provide coding for proper expression of theprotein. From this disclosure an sgRNA that targets the mutation and aCas9 protein can be contacted with an hematopoetic stem cell, and an HDRtemplate introduced, for correction of the mutation for expression ofperoxisomal membrane transporter protein.

In some embodiments, the condition may be neoplasia. In someembodiments, where the condition is neoplasia, the genes to be targetedare any of those listed in Table A (in this case PTEN and so forth). Insome embodiments, the condition may be Age-related Macular Degeneration.In some embodiments, the condition may be a Schizophrenic Disorder. Insome embodiments, the condition may be a Trinucleotide Repeat Disorder.In some embodiments, the condition may be Fragile X Syndrome. In someembodiments, the condition may be a Secretase Related Disorder. In someembodiments, the condition may be a Prion—related disorder. In someembodiments, the condition may be ALS. In some embodiments, thecondition may be a drug addiction. In some embodiments, the conditionmay be Autism. In some embodiments, the condition may be Alzheimer'sDisease. In some embodiments, the condition may be inflammation. In someembodiments, the condition may be Parkinson's Disease.

For example, US Patent Publication No. 20110023145, describes use ofzinc finger nucleases to genetically modify cells, animals and proteinsassociated with autism spectrum disorders (ASD). Autism spectrumdisorders (ASDs) are a group of disorders characterized by qualitativeimpairment in social interaction and communication, and restrictedrepetitive and stereotyped patterns of behavior, interests, andactivities. The three disorders, autism, Asperger syndrome (AS) andpervasive developmental disorder—not otherwise specified (PDD-NOS) are acontinuum of the same disorder with varying degrees of severity,associated intellectual functioning and medical conditions. ASDs arepredominantly genetically determined disorders with a heritability ofaround 90%.

US Patent Publication No. 20110023145 comprises editing of anychromosomal sequences that encode proteins associated with ASD which maybe applied to the CRISPR Cas system of the present invention. Theproteins associated with ASD are typically selected based on anexperimental association of the protein associated with ASD to anincidence or indication of an ASD. For example, the production rate orcirculating concentration of a protein associated with ASD may beelevated or depressed in a population having an ASD relative to apopulation lacking the ASD. Differences in protein levels may beassessed using proteomic techniques including but not limited to Westernblot, immunohistochemical staining, enzyme linked immunosorbent assay(ELISA), and mass spectrometry. Alternatively, the proteins associatedwith ASD may be identified by obtaining gene expression profiles of thegenes encoding the proteins using genomic techniques including but notlimited to DNA microarray analysis, serial analysis of gene expression(SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

Non limiting examples of disease states or disorders that may beassociated with proteins associated with ASD include autism, Aspergersyndrome (AS), pervasive developmental disorder—not otherwise specified(PDD-NOS), Rett's syndrome, tuberous sclerosis, phenylketonuria,Smith-Lemli-Opitz syndrome and fragile X syndrome. By way ofnon-limiting example, proteins associated with ASD include but are notlimited to the following proteins: ATP10C aminophospholipid-MET METreceptor transporting ATPase tyrosine kinase (ATP10C) BZRAP1 MGLUR5(GRM5) Metabotropic glutamate receptor 5 (MGLUR5) CDH10 Cadherin-10MGLUR6 (GRM6) Metabotropic glutamate receptor 6 (MGLUR6) CDH9 Cadherin-9NLGN1 Neuroligin-1 CNTN4 Contactin-4 NLGN2 Neuroligin-2 CNTNAP2Contactin-associated SEMA5A Neuroligin-3 protein-like 2 (CNTNAP2) DHCR77-dehydrocholesterol NLGN4X Neuroligin-4 X-reductase (DHCR7) linkedDOC2A Double C2-like domain-NLGN4Y Neuroligin-4 Y-containing proteinalpha linked DPP6 Dipeptidyl NLGN5 Neuroligin-5 aminopeptidase-likeprotein 6 EN2 engrailed 2 (EN2) NRCAM Neuronal cell adhesion molecule(NRCAM) MDGA2 fragile X mental retardation NRXN1 Neurexin-1 1 (MDGA2)FMR2 (AFF2) AF4/FMR2 family member 2 OR4M2 Olfactory receptor (AFF2) 4M2FOXP2 Forkhead box protein P2 OR4N4 Olfactory receptor (FOXP2) 4N4 FXR1Fragile X mental OXTR oxytocin receptor retardation, autosomal (OXTR)homolog 1 (FXR1) FXR2 Fragile X mental PAH phenylalanine retardation,autosomal hydroxylase (PAH) homolog 2 (FXR2) GABRA1 Gamma-aminobutyricacid PTEN Phosphatase and receptor subunit alpha-1 tensin homologue(GABRA1) (PTEN) GABRA5 GABAA (.gamma.-aminobutyric PTPRZ1 Receptor-typeacid) receptor alpha 5 tyrosine-protein subunit (GABRA5) phosphatasezeta (PTPRZ1) GABRB1 Gamma-aminobutyric acid RELN Reelin receptorsubunit beta-1 (GABRB1) GABRB3 GABAA (.gamma.-aminobutyric RPL10 60Sribosomal acid) receptor .beta.3 subunit protein L10 (GABRB3) GABRG1Gamma-aminobutyric acid SEMA5A Semaphorin-5A receptor subunit gamma-1(SEMA5A) (GABRG1) HIRIP3 HIRA-interacting protein 3 SEZ6L2 seizurerelated 6 homolog (mouse)-like 2 HOXA1 Homeobox protein Hox-A 1 SHANK3SH3 and multiple (HOXA1) ankyrin repeat domains 3 (SHANK3) IL6Interleukin-6 SHBZRAP1 SH3 and multiple ankyrin repeat domains 3(SHBZRAP1) LAMB1 Laminin subunit beta-1 SLC6A4 Serotonin (LAMB1)transporter (SERT) MAPK3 Mitogen-activated protein TAS2R1 Taste receptorkinase 3 type 2 member 1 TAS2R1 MAZ Myc-associated zinc finger TSC1Tuberous sclerosis protein protein 1 MDGA2 MAM domain containing TSC2Tuberous sclerosis glycosylphosphatidylinositol protein 2 anchor 2(MDGA2) MECP2 Methyl CpG binding UBE3A Ubiquitin protein protein 2(MECP2) ligase E3A (UBE3A) MECP2 methyl CpG binding WNT2 Wingless-typeprotein 2 (MECP2) MMTV integration site family, member 2 (WNT2)

The identity of the protein associated with ASD whose chromosomalsequence is edited can and will vary. In preferred embodiments, theproteins associated with ASD whose chromosomal sequence is edited may bethe benzodiazapine receptor (peripheral) associated protein 1 (BZRAP1)encoded by the BZRAP1 gene, the AF4/FMR2 family member 2 protein (AFF2)encoded by the AFF2 gene (also termed MFR2), the fragile X mentalretardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene,the fragile X mental retardation autosomal homolog 2 protein (FXR2)encoded by the FXR2 gene, the MAM domain containingglycosylphosphatidylinositol anchor 2 protein (MDGA2) encoded by theMDGA2 gene, the methyl CpG binding protein 2 (MECP2) encoded by theMECP2 gene, the metabotropic glutamate receptor 5 (MGLUR5) encoded bythe MGLUR5-1 gene (also termed GRM5), the neurexin 1 protein encoded bythe NRXN1 gene, or the semaphorin-5A protein (SEMA5A) encoded by theSEMA5A gene. In an exemplary embodiment, the genetically modified animalis a rat, and the edited chromosomal sequence encoding the proteinassociated with ASD is as listed below: BZRAP1 benzodiazapine receptorXM_002727789, (peripheral) associated XM_213427, protein 1 (BZRAP1)XM_002724533, XM_001081125 AFF2 (FMR2) AF4/FMR2 family member 2XM_219832, (AFF2) XM_001054673 FXR1 Fragile X mental NM_001012179retardation, autosomal homolog 1 (FXR1) FXR2 Fragile X mentalNM_001100647 retardation, autosomal homolog 2 (FXR2) MDGA2 MAM domaincontaining NM_199269 glycosylphosphatidylinositol anchor 2 (MDGA2) MECP2Methyl CpG binding NM_022673 protein 2 (MECP2) MGLUR5 Metabotropicglutamate NM_017012 (GRM5) receptor 5 (MGLUR5) NRXN1 Neurexin-1NM_021767 SEMA5A Semaphorin-5A (SEMA5A) NM_001107659

Exemplary animals or cells may comprise one, two, three, four, five,six, seven, eight, or nine or more inactivated chromosomal sequencesencoding a protein associated with ASD, and zero, one, two, three, four,five, six, seven, eight, nine or more chromosomally integrated sequencesencoding proteins associated with ASD. The edited or integratedchromosomal sequence may be modified to encode an altered proteinassociated with ASD. Non-limiting examples of mutations in proteinsassociated with ASD include the L18Q mutation in neurexin 1 where theleucine at position 18 is replaced with a glutamine, the R451C mutationin neuroligin 3 where the arginine at position 451 is replaced with acysteine, the R87W mutation in neuroligin 4 where the arginine atposition 87 is replaced with a tryptophan, and the I425V mutation inserotonin transporter where the isoleucine at position 425 is replacedwith a valine. A number of other mutations and chromosomalrearrangements in ASD-related chromosomal sequences have been associatedwith ASD and are known in the art. See, for example, Freitag et al.(2010) Eur. Child. Adolesc. Psychiatry 19:169-178, and Bucan et al.(2009) PLoS Genetics 5: e1000536, the disclosure of which isincorporated by reference herein in its entirety. Examples of proteinsassociated with Parkinson's disease include but are not limited toα-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1, Synphilin-1, and NURRI.Examples of addiction-related proteins may include ABAT for example.Examples of inflammation-related proteins may include the monocytechemoattractant protein-1 (MCP1) encoded by the Ccr2 gene, the C-Cchemokine receptor type 5 (CCR5) encoded by the Ccr5 gene, the IgGreceptor IIB (FCGR2b, also termed CD32) encoded by the Fcgr2b gene, orthe Fc epsilon R1g (FCER1g) protein encoded by the Fcer1g gene, forexample. Examples of cardiovascular diseases associated proteins mayinclude IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53(tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin) synthase),MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8(ATP-binding cassette, sub-family G (WHITE), member 8), or CTSK(cathepsin K), for example. For example, US Patent Publication No.20110023153, describes use of zinc finger nucleases to geneticallymodify cells, animals and proteins associated with Alzheimer's Disease.Once modified cells and animals may be further tested using knownmethods to study the effects of the targeted mutations on thedevelopment and/or progression of AD using measures commonly used in thestudy of AD—such as, without limitation, learning and memory, anxiety,depression, addiction, and sensory motor functions as well as assaysthat measure behavioral, functional, pathological, metaboloic andbiochemical function.

The present invention comprises editing of any chromosomal sequencesthat encode proteins associated with AD. The AD-related proteins aretypically selected based on an experimental association of theAD-related protein to an AD disorder. For example, the production rateor circulating concentration of an AD-related protein may be elevated ordepressed in a population having an AD disorder relative to a populationlacking the AD disorder. Differences in protein levels may be assessedusing proteomic techniques including but not limited to Western blot,immunohistochemical staining, enzyme linked immunosorbent assay (ELISA),and mass spectrometry. Alternatively, the AD-related proteins may beidentified by obtaining gene expression profiles of the genes encodingthe proteins using genomic techniques including but not limited to DNAmicroarray analysis, serial analysis of gene expression (SAGE), andquantitative real-time polymerase chain reaction (Q-PCR). Examples ofAlzheimer's disease associated proteins may include the very low densitylipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, theubiquitin-like modifier activating enzyme 1 (UBA1) encoded by the UBA1gene, or the NEDD8-activating enzyme E1 catalytic subunit protein(UBE1C) encoded by the UBA3 gene, for example. By way of non-limitingexample, proteins associated with AD include but are not limited to theproteins listed as follows: Chromosomal Sequence Encoded Protein ALAS2Delta-aminolevulinate synthase 2 (ALAS2) ABCA1 ATP-binding cassettetransporter (ABCA1) ACE Angiotensin I-converting enzyme (ACE) APOEApolipoprotein E precursor (APOE) APP amyloid precursor protein (APP)AQP1 aquaporin 1 protein (AQP1) BIN1 Myc box-dependent-interactingprotein 1 or bridging integrator 1 protein (BIN1) BDNF brain-derivedneurotrophic factor (BDNF) BTNL8 Butyrophilin-like protein 8 (BTNL8)ClORF49 chromosome 1 open reading frame 49 CDH4 Cadherin-4 CHRNB2Neuronal acetylcholine receptor subunit beta-2 CKLFSF2 CKLF-like MARVELtransmembrane domain-containing protein 2 (CKLFSF2) CLEC4E C-type lectindomain family 4, member e (CLEC4E) CLU clusterin protein (also known asapoplipoprotein J) CR1 Erythrocyte complement receptor 1 (CR1, alsoknown as CD35, C3b/C4b receptor and immune adherence receptor) CR1LErythrocyte complement receptor 1 (CR1L) CSF3R granulocytecolony-stimulating factor 3 receptor (CSF3R) CST3 Cystatin C or cystatin3 CYP2C Cytochrome P450 2C DAPK1 Death-associated protein kinase 1(DAPK1) ESR1 Estrogen receptor 1 FCAR Fc fragment of IgA receptor (FCAR,also known as CD89) FCGR3B Fc fragment of IgG, low affinity IIb,receptor (FCGR3B or CD16b) FFA2 Free fatty acid receptor 2 (FFA2) FGAFibrinogen (Factor I) GAB2 GRB2-associated-binding protein 2 (GAB2) GAB2GRB2-associated-binding protein 2 (GAB2) GALP Galanin-like peptideGAPDHS Glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS)GMPB GMBP HP Haptoglobin (HP) HTR7 5-hydroxytryptamine (serotonin)receptor 7 (adenylate cyclase-coupled) IDE Insulin degrading enzymeIF127 IF127 IFI6 Interferon, alpha-inducible protein 6 (IFI6) IFIT2Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2)IL1RN interleukin-1 receptor antagonist (IL-1RA) IL8RA Interleukin 8receptor, alpha (IL8RA or CD181) IL8RB Interleukin 8 receptor, beta(IL8RB) JAG1 Jagged 1 (JAG1) KCNJ15 Potassium inwardly-rectifyingchannel, subfamily J, member 15 (KCNJ15) LRP6 Low-density lipoproteinreceptor-related protein 6 (LRP6) MAPT microtubule-associated proteintau (MAPT) MARK4 MAP/microtubule affinity-regulating kinase 4 (MARK4)MPHOSPH1 M-phase phosphoprotein 1 MTHFR 5,10-methylenetetrahydrofolatereductase MX2 Interferon-induced GTP-binding protein Mx2 NBN Nibrin,also known as NBN NCSTN Nicastrin NIACR2 Niacin receptor 2 (NIACR2, alsoknown as GPR109B) NMNAT3 nicotinamide nucleotide adenylyltransferase 3NTM Neurotrimin (or HNT) ORM1 Orosmucoid 1 (ORM1) or Alpha-1-acidglycoprotein 1 P2RY13 P2Y purinoceptor 13 (P2RY13) PBEF1 Nicotinamidephosphoribosyltransferase (NAmPRTase or Nampt) also known as pre-B-cellcolony-enhancing factor 1 (PBEF1) or visfatin PCK1 Phosphoenolpyruvatecarboxykinase PICALM phosphatidylinositol binding clathrin assemblyprotein (PICALM) PLAU Urokinase-type plasminogen activator (PLAU) PLXNC1Plexin C1 (PLXNC1) PRNP Prion protein PSEN1 presenilin 1 protein (PSEN1)PSEN2 presenilin 2 protein (PSEN2) PTPRA protein tyrosine phosphatasereceptor type A protein (PTPRA) RALGPS2 Ral GEF with PH domain and SH3binding motif 2 (RALGPS2) RGSL2 regulator of G-protein signaling like 2(RGSL2) SELENBP1 Selenium binding protein 1 (SELNBP1) SLC25A37Mitoferrin-1 SORL1 sortilin-related receptor L(DLR class) Arepeats-containing protein (SORL1) TF Transferrin TFAM Mitochondrialtranscription factor A TNF Tumor necrosis factor TNFRSF10C Tumornecrosis factor receptor superfamily member 10C (TNFRSF10C) TNFSF10Tumor necrosis factor receptor superfamily, (TRAIL) member 10a (TNFSF10)UBA1 ubiquitin-like modifier activating enzyme 1 (UBA1) UBA3NEDD8-activating enzyme E1 catalytic subunit protein (UBE1C) UBBubiquitin B protein (UBB) UBQLN1 Ubiquilin-1 UCHL1 ubiquitincarboxyl-terminal esterase L1 protein (UCHL1) UCHL3 ubiquitincarboxyl-terminal hydrolase isozyme L3 protein (UCHL3) VLDLR very lowdensity lipoprotein receptor protein (VLDLR). In exemplary embodiments,the proteins associated with AD whose chromosomal sequence is edited maybe the very low density lipoprotein receptor protein (VLDLR) encoded bythe VLDLR gene, the ubiquitin-like modifier activating enzyme 1 (UBA1)encoded by the UBA1 gene, the NEDD8-activating enzyme E1 catalyticsubunit protein (UBE1C) encoded by the UBA3 gene, the aquaporin 1protein (AQP1) encoded by the AQP1 gene, the ubiquitin carboxyl-terminalesterase L1 protein (UCHL1) encoded by the UCHL1 gene, the ubiquitincarboxyl-terminal hydrolase isozyme L3 protein (UCHL3) encoded by theUCHL3 gene, the ubiquitin B protein (UBB) encoded by the UBB gene, themicrotubule-associated protein tau (MAPT) encoded by the MAPT gene, theprotein tyrosine phosphatase receptor type A protein (PTPRA) encoded bythe PTPRA gene, the phosphatidylinositol binding clathrin assemblyprotein (PICALM) encoded by the PICALM gene, the clusterin protein (alsoknown as apoplipoprotein J) encoded by the CLU gene, the presenilin 1protein encoded by the PSEN1 gene, the presenilin 2 protein encoded bythe PSEN2 gene, the sortilin-related receptor L(DLR class) Arepeats-containing protein (SORL1) protein encoded by the SORL1 gene,the amyloid precursor protein (APP) encoded by the APP gene, theApolipoprotein E precursor (APOE) encoded by the APOE gene, or thebrain-derived neurotrophic factor (BDNF) encoded by the BDNF gene. In anexemplary embodiment, the genetically modified animal is a rat, and theedited chromosomal sequence encoding the protein associated with AD isas as follows: APP amyloid precursor protein (APP) NM_019288 AQP1aquaporin 1 protein (AQP1) NM_012778 BDNF Brain-derived neurotrophicfactor NM_012513 CLU clusterin protein (also known as NM_053021apoplipoprotein J) MAPT microtubule-associated protein NM_017212 tau(MAPT) PICALM phosphatidylinositol binding NM_053554 clathrin assemblyprotein (PICALM) PSEN1 presenilin 1 protein (PSEN1) NM_019163 PSEN2presenilin 2 protein (PSEN2) NM_031087 PTPRA protein tyrosinephosphatase NM_012763 receptor type A protein (PTPRA) SORL1sortilin-related receptor L(DLR NM_053519, class) A repeats-containingXM_001065506, protein (SORL1) XM_217115 UBA1 ubiquitin-like modifieractivating NM_001014080 enzyme 1 (UBA1) UBA3 NEDD8-activating enzyme E1NM_057205 catalytic subunit protein (UBEIC) UBB ubiquitin B protein(UBB) NM_138895 UCHL1 ubiquitin carboxyl-terminal NM_017237 esterase L1protein (UCHL1) UCHL3 ubiquitin carboxyl-terminal NM_001110165 hydrolaseisozyme L3 protein (UCHL3) VLDLR very low density lipoprotein NM_013155receptor protein (VLDLR). The animal or cell may comprise 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more disrupted chromosomalsequences encoding a protein associated with AD and zero, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more chromosomally integratedsequences encoding a protein associated with AD. The edited orintegrated chromosomal sequence may be modified to encode an alteredprotein associated with AD. A number of mutations in AD-relatedchromosomal sequences have been associated with AD. For instance, theV7171 (i.e. valine at position 717 is changed to isoleucine) missensemutation in APP causes familial AD. Multiple mutations in thepresenilin-1 protein, such as H163R (i.e. histidine at position 163 ischanged to arginine), A246E (i.e. alanine at position 246 is changed toglutamate), L286V (i.e. leucine at position 286 is changed to valine)and C410Y (i.e. cysteine at position 410 is changed to tyrosine) causefamilial Alzheimer's type 3. Mutations in the presenilin-2 protein, suchas N141 I (i.e. asparagine at position 141 is changed to isoleucine),M239V (i.e. methionine at position 239 is changed to valine), and D439A(i.e. aspartate at position 439 is changed to alanine) cause familialAlzheimer's type 4. Other associations of genetic variants inAD-associated genes and disease are known in the art. See, for example,Waring et al. (2008) Arch. Neurol. 65:329-334, the disclosure of whichis incorporated by reference herein in its entirety.

Examples of proteins associated Autism Spectrum Disorder may include thebenzodiazapine receptor (peripheral) associated protein 1 (BZRAP1)encoded by the BZRAP1 gene, the AF4/FMR2 family member 2 protein (AFF2)encoded by the AFF2 gene (also termed MFR2), the fragile X mentalretardation autosomal homolog 1 protein (FXR1) encoded by the FXR1 gene,or the fragile X mental retardation autosomal homolog 2 protein (FXR2)encoded by the FXR2 gene, for example.

Examples of proteins associated Macular Degeneration may include theATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4)encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded bythe APOE gene, or the chemokine (C-C motif) Ligand 2 protein (CCL2)encoded by the CCL2 gene, for example.

Examples of proteins associated Schizophrenia may include NRG1, ErbB4,CPLX1, TPH1, TPH2, NRXN1, GSK3A, BDNF, DISC1, GSK3B, and combinationsthereof.

Examples of proteins involved in tumor suppression may include ATM(ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3related), EGFR (epidermal growth factor receptor), ERBB2 (v-erb-b2erythroblastic leukemia viral oncogene homolog 2), ERBB3 (v-erb-b2erythroblastic leukemia viral oncogene homolog 3), ERBB4 (v-erb-b2erythroblastic leukemia viral oncogene homolog 4), Notch 1, Notch2,Notch 3, or Notch 4, for example.

Examples of proteins associated with a secretase disorder may includePSENEN (presenilin enhancer 2 homolog (C. elegans)), CTSB (cathepsin B),PSEN1 (presenilin 1), APP (amyloid beta (A4) precursor protein), APH1B(anterior pharynx defective 1 homolog B (C. elegans)), PSEN2 (presenilin2 (Alzheimer disease 4)), or BACE1 (beta-site APP-cleaving enzyme 1),for example. For example, US Patent Publication No. 20110023146,describes use of zinc finger nucleases to genetically modify cells,animals and proteins associated with secretase-associated disorders.Secretases are essential for processing pre-proteins into theirbiologically active forms. Defects in various components of thesecretase pathways contribute to many disorders, particularly those withhallmark amyloidogenesis or amyloid plaques, such as Alzheimer's disease(AD).

A secretase disorder and the proteins associated with these disordersare a diverse set of proteins that effect susceptibility for numerousdisorders, the presence of the disorder, the severity of the disorder,or any combination thereof. The present disclosure comprises editing ofany chromosomal sequences that encode proteins associated with asecretase disorder. The proteins associated with a secretase disorderare typically selected based on an experimental association of thesecretase-related proteins with the development of a secretase disorder.For example, the production rate or circulating concentration of aprotein associated with a secretase disorder may be elevated ordepressed in a population with a secretase disorder relative to apopulation without a secretase disorder. Differences in protein levelsmay be assessed using proteomic techniques including but not limited toWestern blot, immunohistochemical staining, enzyme linked immunosorbentassay (ELISA), and mass spectrometry. Alternatively, the proteinassociated with a secretase disorder may be identified by obtaining geneexpression profiles of the genes encoding the proteins using genomictechniques including but not limited to DNA microarray analysis, serialanalysis of gene expression (SAGE), and quantitative real-timepolymerase chain reaction (Q-PCR). By way of non-limiting example,proteins associated with a secretase disorder include PSENEN (presenilinenhancer 2 homolog (C. elegans)), CTSB (cathepsin B), PSEN1 (presenilin1), APP (amyloid beta (A4) precursor protein), APH1B (anterior pharynxdefective 1 homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimerdisease 4)), BACE1 (beta-site APP-cleaving enzyme 1), ITM2B (integralmembrane protein 2B), CTSD (cathepsin D), NOTCH1 (Notch homolog 1,translocation-associated (Drosophila)), TNF (tumor necrosis factor (TNFsuperfamily, member 2)), INS (insulin), DYT10 (dystonia 10), ADAM17(ADAM metallopeptidase domain 17), APOE (apolipoprotein E), ACE(angiotensin I converting enzyme (peptidyl-dipeptidase A) 1), STN(statin), TP53 (tumor protein p53), IL6 (interleukin 6 (interferon, beta2)), NGFR (nerve growth factor receptor (TNFR superfamily, member 16)),IL1B (interleukin 1, beta), ACHE (acetylcholinesterase (Yt bloodgroup)), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88 kDa),IGF1 (insulin-like growth factor 1 (somatomedin C)), IFNG (interferon,gamma), NRG1 (neuregulin 1), CASP3 (caspase 3, apoptosis-relatedcysteine peptidase), MAPK1 (mitogen-activated protein kinase 1), CDH1(cadherin 1, type 1, E-cadherin (epithelial)), APBB1 (amyloid beta (A4)precursor protein-binding, family B, member 1 (Fe65)), HMGCR(3-hydroxy-3-methylglutaryl-Coenzyme A reductase), CREB1 (cAMPresponsive element binding protein 1), PTGS2 (prostaglandin-endoperoxidesynthase 2 (prostaglandin G/H synthase and cyclooxygenase)), HES1 (hairyand enhancer of split 1, (Drosophila)), CAT (catalase), TGFB1(transforming growth factor, beta 1), ENO2 (enolase 2 (gamma,neuronal)), ERBB4 (v-erb-a erythroblastic leukemia viral oncogenehomolog 4 (avian)), TRAPPC10 (trafficking protein particle complex 10),MAOB (monoamine oxidase B), NGF (nerve growth factor (betapolypeptide)), MMP12 (matrix metallopeptidase 12 (macrophage elastase)),JAG1 (jagged 1 (Alagille syndrome)), CD40LG (CD40 ligand), PPARG(peroxisome proliferator-activated receptor gamma), FGF2 (fibroblastgrowth factor 2 (basic)), IL3 (interleukin 3 (colony-stimulating factor,multiple)), LRP1 (low density lipoprotein receptor-related protein 1),NOTCH4 (Notch homolog 4 (Drosophila)), MAPK8 (mitogen-activated proteinkinase 8), PREP (prolyl endopeptidase), NOTCH3 (Notch homolog 3(Drosophila)), PRNP (prion protein), CTSG (cathepsin G), EGF (epidermalgrowth factor (beta-urogastrone)), REN (renin), CD44 (CD44 molecule(Indian blood group)), SELP (selectin P (granule membrane protein 140kDa, antigen CD62)), GHR (growth hormone receptor), ADCYAP1 (adenylatecyclase activating polypeptide 1 (pituitary)), INSR (insulin receptor),GFAP (glial fibrillary acidic protein), MMP3 (matrix metallopeptidase 3(stromelysin 1, progelatinase)), MAPK10 (mitogen-activated proteinkinase 10), SP1 (Sp1 transcription factor), MYC (v-myc myelocytomatosisviral oncogene homolog (avian)), CTSE (cathepsin E), PPARA (peroxisomeproliferator-activated receptor alpha), JUN (jun oncogene), TIMP1 (TIMPmetallopeptidase inhibitor 1), IL5 (interleukin 5 (colony-stimulatingfactor, eosinophil)), IL1A (interleukin 1, alpha), MMP9 (matrixmetallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IVcollagenase)), HTR4 (5-hydroxytryptamine (serotonin) receptor 4), HSPG2(heparan sulfate proteoglycan 2), KRAS (v-Ki-ras2 Kirsten rat sarcomaviral oncogene homolog), CYCS (cytochrome c, somatic), SMG1 (SMG1homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans)),IL1R1 (interleukin 1 receptor, type I), PROK (prokineticin 1), MAPK3(mitogen-activated protein kinase 3), NTRK1 (neurotrophic tyrosinekinase, receptor, type 1), IL13 (interleukin 13), MME (membranemetallo-endopeptidase), TKT (transketolase), CXCR2 (chemokine (C-X-Cmotif) receptor 2), IGF1R (insulin-like growth factor 1 receptor), RARA(retinoic acid receptor, alpha), CREBBP (CREB binding protein), PTGS1(prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase andcyclooxygenase)), GALT (galactose-1-phosphate uridylyltransferase),CHRM1 (cholinergic receptor, muscarinic 1), ATXN1 (ataxin 1), PAWR(PRKC, apoptosis, WT1, regulator), NOTCH2 (Notch homolog 2(Drosophila)), M6PR (mannose-6-phosphate receptor (cation dependent)),CYP46A 1 (cytochrome P450, family 46, subfamily A, polypeptide 1), CSNK1D (casein kinase 1, delta), MAPK14 (mitogen-activated protein kinase14), PRG2 (proteoglycan 2, bone marrow (natural killer cell activator,eosinophil granule major basic protein)), PRKCA (protein kinase C,alpha), L1 CAM (L1 cell adhesion molecule), CD40 (CD40 molecule, TNFreceptor superfamily member 5), NR1I2 (nuclear receptor subfamily 1,group I, member 2), JAG2 (jagged 2), CTNND1 (catenin(cadherin-associated protein), delta 1), CDH2 (cadherin 2, type 1,N-cadherin (neuronal)), CMA1 (chymase 1, mast cell), SORT1 (sortilin 1),DLK1 (delta-like 1 homolog (Drosophila)), THEM4 (thioesterasesuperfamily member 4), JUP (junction plakoglobin), CD46 (CD46 molecule,complement regulatory protein), CCL11 (chemokine (C-C motif) ligand 11),CAV3 (caveolin 3), RNASE3 (ribonuclease, RNase A family, 3 (eosinophilcationic protein)), HSPA8 (heat shock 70 kDa protein 8), CASP9 (caspase9, apoptosis-related cysteine peptidase), CYP3A4 (cytochrome P450,family 3, subfamily A, polypeptide 4), CCR3 (chemokine (C-C motif)receptor 3), TFAP2A (transcription factor AP-2 alpha (activatingenhancer binding protein 2 alpha)), SCP2 (sterol carrier protein 2),CDK4 (cyclin-dependent kinase 4), HIF1A (hypoxia inducible factor 1,alpha subunit (basic helix-loop-helix transcription factor)), TCF7L2(transcription factor 7-like 2 (T-cell specific, HMG-box)), IL1R2(interleukin 1 receptor, type II), B3GALTL (beta1,3-galactosyltransferase-like), MDM2 (Mdm2 p53 binding protein homolog(mouse)), RELA (v-rel reticuloendotheliosis viral oncogene homolog A(avian)), CASP7 (caspase 7, apoptosis-related cysteine peptidase), IDE(insulin-degrading enzyme), FABP4 (fatty acid binding protein 4,adipocyte), CASK (calcium/calmodulin-dependent serine protein kinase(MAGUK family)), ADCYAPIR1 (adenylate cyclase activating polypeptide 1(pituitary) receptor type I), ATF4 (activating transcription factor 4(tax-responsive enhancer element B67)), PDGFA (platelet-derived growthfactor alpha polypeptide), C21 or f33 (chromosome 21 open reading frame33), SCG5 (secretogranin V (7B2 protein)), RNF123 (ring finger protein123), NFKB1 (nuclear factor of kappa light polypeptide gene enhancer inB-cells 1), ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogenehomolog 2, neuro/glioblastoma derived oncogene homolog (avian)), CAV1(caveolin 1, caveolae protein, 22 kDa), MMP7 (matrix metallopeptidase 7(matrilysin, uterine)), TGFA (transforming growth factor, alpha), RXRA(retinoid X receptor, alpha), STX1A (syntaxin 1A (brain)), PSMC4(proteasome (prosome, macropain) 26S subunit, ATPase, 4), P2RY2(purinergic receptor P2Y, G-protein coupled, 2), TNFRSF21 (tumornecrosis factor receptor superfamily, member 21), DLG1 (discs, largehomolog 1 (Drosophila)), NUMBL (numb homolog (Drosophila)-like), SPN(sialophorin), PLSCR1 (phospholipid scramblase 1), UBQLN2 (ubiquilin 2),UBQLN1 (ubiquilin 1), PCSK7 (proprotein convertase subtilisin/kexin type7), SPON1 (spondin 1, extracellular matrix protein), SILV (silverhomolog (mouse)), QPCT (glutaminyl-peptide cyclotransferase), HESS(hairy and enhancer of split 5 (Drosophila)), GCC1 (GRIP and coiled-coildomain containing 1), and any combination thereof. The geneticallymodified animal or cell may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore disrupted chromosomal sequences encoding a protein associated witha secretase disorder and zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morechromosomally integrated sequences encoding a disrupted proteinassociated with a secretase disorder.

Examples of proteins associated with Amyotrophic Lateral Sclerosis mayinclude SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateralsclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA binding protein),VAGFA (vascular endothelial growth factor A), VAGFB (vascularendothelial growth factor B), and VAGFC (vascular endothelial growthfactor C), and any combination thereof. For example, US PatentPublication No. 20110023144, describes use of zinc finger nucleases togenetically modify cells, animals and proteins associated withamyotrophyic lateral sclerosis (ALS) disease. ALS is characterized bythe gradual steady degeneration of certain nerve cells in the braincortex, brain stem, and spinal cord involved in voluntary movement.

Motor neuron disorders and the proteins associated with these disordersare a diverse set of proteins that effect susceptibility for developinga motor neuron disorder, the presence of the motor neuron disorder, theseverity of the motor neuron disorder or any combination thereof. Thepresent disclosure comprises editing of any chromosomal sequences thatencode proteins associated with ALS disease, a specific motor neurondisorder. The proteins associated with ALS are typically selected basedon an experimental association of ALS-related proteins to ALS. Forexample, the production rate or circulating concentration of a proteinassociated with ALS may be elevated or depressed in a population withALS relative to a population without ALS. Differences in protein levelsmay be assessed using proteomic techniques including but not limited toWestern blot, immunohistochemical staining, enzyme linked immunosorbentassay (ELISA), and mass spectrometry. Alternatively, the proteinsassociated with ALS may be identified by obtaining gene expressionprofiles of the genes encoding the proteins using genomic techniquesincluding but not limited to DNA microarray analysis, serial analysis ofgene expression (SAGE), and quantitative real-time polymerase chainreaction (Q-PCR). By way of non-limiting example, proteins associatedwith ALS include but are not limited to the following proteins: SOD1superoxide dismutase 1, ALS3 amyotrophic lateral soluble sclerosis 3SETX senataxin ALS5 amyotrophic lateral sclerosis 5 FUS fused in sarcomaALS7 amyotrophic lateral sclerosis 7 ALS2 amyotrophic lateral DPP6Dipeptidyl-peptidase 6 sclerosis 2 NEFH neurofilament, heavy PTGS1prostaglandin-polypeptide endoperoxide synthase 1 SLC1A2 solute carrierfamily 1 TNFRSF10B tumor necrosis factor (glial high affinity receptorsuperfamily, glutamate transporter), member 10b member 2 PRPH peripherinHSP90AA1 heat shock protein 90 kDa alpha (cytosolic), class A member 1GRIA2 glutamate receptor, IFNG interferon, gamma ionotropic, AMPA 2S100B S100 calcium binding FGF2 fibroblast growth factor 2 protein BAOX1 aldehyde oxidase 1 CS citrate synthase TARDBP TAR DNA bindingprotein TXN thioredoxin RAPH1 Ras association MAP3K5 mitogen-activatedprotein (RaIGDS/AF-6) and kinase 5 pleckstrin homology domains 1 NBEAL1neurobeachin-like 1 GPX1 glutathione peroxidase 1 ICA1L islet cellautoantigen RAC1 ras-related C3 botulinum 1.69 kDa-like toxin substrate1 MAPT microtubule-associated ITPR2 inositol 1,4,5-protein tautriphosphate receptor, type 2 ALS2CR4 amyotrophic lateral GLSglutaminase sclerosis 2 (juvenile) chromosome region, candidate 4ALS2CR8 amyotrophic lateral CNTFR ciliary neurotrophic factor sclerosis2 (juvenile) receptor chromosome region, candidate 8 ALS2CR11amyotrophic lateral FOLH1 folate hydrolase 1 sclerosis 2 (juvenile)chromosome region, candidate 11 FAM117B family with sequence P4HB prolyl4-hydroxylase, similarity 117, member B beta polypeptide CNTF ciliaryneurotrophic factor SQSTM1 sequestosome 1 STRADB STE20-related kinaseNAIP NLR family, apoptosis adaptor beta inhibitory protein YWHAQtyrosine 3-SLC33A1 solute carrier family 33 monooxygenase/tryptoph(acetyl-CoA transporter), an 5-monooxygenase member 1 activationprotein, theta polypeptide TRAK2 trafficking protein, FIG. 4 FIG. 4homolog, SACI kinesin binding 2 lipid phosphatase domain containingNIF3L1 NIF3 NGG1 interacting INA internexin neuronal factor 3-like 1intermediate filament protein, alpha PARD3B par-3 partitioning COX8Acytochrome c oxidase defective 3 homolog B subunit VIIIA CDK15cyclin-dependent kinase HECW1 HECT, C2 and WW 15 domain containing E3ubiquitin protein ligase 1 NOS1 nitric oxide synthase 1 MET metproto-oncogene SOD2 superoxide dismutase 2, HSPB1 heat shock 27 kDamitochondrial protein 1 NEFL neurofilament, light CTSB cathepsin Bpolypeptide ANG angiogenin, HSPA8 heat shock 70 kDa ribonuclease, RNaseA protein 8 family, 5 VAPB VAMP (vesicle-ESR1 estrogen receptor 1associated membrane protein)-associated protein B and C SNCA synuclein,alpha HGF hepatocyte growth factor CAT catalase ACTB actin, beta NEFMneurofilament, medium TH tyrosine hydroxylase polypeptide BCL2 B-cellCLL/lymphoma 2 FAS Fas (TNF receptor superfamily, member 6) CASP3caspase 3, apoptosis-CLU clusterin related cysteine peptidase SMN1survival of motor neuron G6PD glucose-6-phosphate 1, telomericdehydrogenase BAX BCL2-associated X HSF1 heat shock transcriptionprotein factor 1 RNF19A ring finger protein 19A JUN jun oncogeneALS2CR12 amyotrophic lateral HSPA5 heat shock 70 kDa sclerosis 2(juvenile) protein 5 chromosome region, candidate 12 MAPK14mitogen-activated protein IL10 interleukin 10 kinase 14 APEX1 APEXnuclease TXNRD1 thioredoxin reductase 1 (multifunctional DNA repairenzyme) 1 NOS2 nitric oxide synthase 2, TIMP1 TIMP metallopeptidaseinducible inhibitor 1 CASP9 caspase 9, apoptosis-XIAP X-linked inhibitorof related cysteine apoptosis peptidase GLG1 golgi glycoprotein 1 EPOerythropoietin VEGFA vascular endothelial ELN elastin growth factor AGDNF glial cell derived NFE2L2 nuclear factor (erythroid-neurotrophicfactor derived 2)-like 2 SLC6A3 solute carrier family 6 HSPA4 heat shock70 kDa (neurotransmitter protein 4 transporter, dopamine), member 3 APOEapolipoprotein E PSMB8 proteasome (prosome, macropain) subunit, betatype, 8 DCTN1 dynactin 1 TIMP3 TIMP metallopeptidase inhibitor 3 KIFAP3kinesin-associated SLC1A1 solute carrier family 1 protein 3(neuronal/epithelial high affinity glutamate transporter, system Xag),member 1 SMN2 survival of motor neuron CCNC cyclin C 2, centromeric MPP4membrane protein, STUB1 STIP1 homology and U-palmitoylated 4 boxcontaining protein 1 ALS2 amyloid beta (A4) PRDX6 peroxiredoxin 6precursor protein SYP synaptophysin CABIN1 calcineurin binding protein 1CASP1 caspase 1, apoptosis-GART phosphoribosylglycinami related cysteinede formyltransferase, peptidase phosphoribosylglycinami de synthetase,phosphoribosylaminoimi dazole synthetase CDK5 cyclin-dependent kinase 5ATXN3 ataxin 3 RTN4 reticulon 4 C1QB complement component 1, qsubcomponent, B chain VEGFC nerve growth factor HTT huntingtin receptorPARK7 Parkinson disease 7 XDH xanthine dehydrogenase GFAP glialfibrillary acidic MAP2 microtubule-associated protein protein 2 CYCScytochrome c, somatic FCGR3B Fc fragment of IgG, low affinity IIIb, CCScopper chaperone for UBL5 ubiquitin-like 5 superoxide dismutase MMP9matrix metallopeptidase SLC18A3 solute carrier family 18 9 ((vesicularacetylcholine), member 3 TRPM7 transient receptor HSPB2 heat shock 27kDa potential cation channel, protein 2 subfamily M, member 7 AKT1 v-aktmurine thymoma DERL1 Der1-like domain family, viral oncogene homolog 1member 1 CCL2 chemokine (C-C motif) NGRN neugrin, neurite ligand 2outgrowth associated GSR glutathione reductase TPPP3 tubulinpolymerization-promoting protein family member 3 APAF1 apoptoticpeptidase BTBD10 BTB (POZ) domain activating factor 1 containing 10GLUD1 glutamate CXCR4 chemokine (C-X-C motif) dehydrogenase 1 receptor 4SLC1A3 solute carrier family 1 FLT1 fms-related tyrosine (glial highaffinity glutamate transporter), member 3 kinase 1 PON1 paraoxonase 1 ARandrogen receptor LIF leukemia inhibitory factor ERBB3 v-erb-b2erythroblastic leukemia viral oncogene homolog 3 LGALSI lectin,galactoside-CD44 CD44 molecule binding, soluble, 1 TP53 tumor proteinp53 TLR3 toll-like receptor 3 GRIA1 glutamate receptor, GAPDHglyceraldehyde-3-ionotropic, AMPA 1 phosphate dehydrogenase GRIK1glutamate receptor, DES desmin ionotropic, kainate 1 CHAT cholineacetyltransferase FLT4 fins-related tyrosine kinase 4 CHMP2B chromatinmodifying BAG1 BCL2-associated protein 2B athanogene MT3 metallothionein3 CHRNA4 cholinergic receptor, nicotinic, alpha 4 GSS glutathionesynthetase BAK1 BCL2-antagonist/killer 1 KDR kinase insert domain GSTP1glutathione S-transferase receptor (a type III pi 1 receptor tyrosinekinase) OGG1 8-oxoguanine DNA IL6 interleukin 6 (interferon, glycosylasebeta 2). The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more disrupted chromosomal sequences encoding a protein associatedwith ALS and zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomallyintegrated sequences encoding the disrupted protein associated with ALS.Preferred proteins associated with ALS include SOD1 (superoxidedismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused insarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelialgrowth factor A), VAGFB (vascular endothelial growth factor B), andVAGFC (vascular endothelial growth factor C), and any combinationthereof.

Examples of proteins associated with prion diseases may include SOD1(superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS(fused in sarcoma), TARDBP (TAR DNA binding protein), VAGFA (vascularendothelial growth factor A), VAGFB (vascular endothelial growth factorB), and VAGFC (vascular endothelial growth factor C), and anycombination thereof.

Examples of proteins related to neurodegenerative conditions in priondisorders may include A2M (Alpha-2-Macroglobulin), AATF (Apoptosisantagonizing transcription factor), ACPP (Acid phosphatase prostate),ACTA2 (Actin alpha 2 smooth muscle aorta), ADAM22 (ADAM metallopeptidasedomain), ADORA3 (Adenosine A3 receptor), or ADRA1D (Alpha-1D adrenergicreceptor for Alpha-1D adrenoreceptor), for example.

Examples of proteins associated with Immunodeficiency may include A2M[alpha-2-macroglobulin]; AANAT [arylalkylamine N-acetyltransferase];ABCA1 [ATP-binding cassette, sub-family A (ABC1), member 1]; ABCA2[ATP-binding cassette, sub-family A (ABC1), member 2]; or ABCA3[ATP-binding cassette, sub-family A (ABC1), member 3]; for example.

Examples of proteins associated with Trinucleotide Repeat Disordersinclude AR (androgen receptor), FMR1 (fragile X mental retardation 1),HTT (huntingtin), or DMPK (dystrophia myotonica-protein kinase), FXN(frataxin), ATXN2 (ataxin 2), for example. Examples of proteinsassociated with Neurotransmission Disorders include SST (somatostatin),NOS1 (nitric oxide synthase 1 (neuronal)), ADRA2A (adrenergic,alpha-2A-, receptor), ADRA2C (adrenergic, alpha-2C-, receptor), TACR1(tachykinin receptor 1), or HTR2c (5-hydroxytryptamine (serotonin)receptor 2C), for example. Examples of neurodevelopmental-associatedsequences include A2BPI [ataxin 2-binding protein 1], AADAT[aminoadipate aminotransferase], AANAT [arylalkylamineN-acetyltransferase], ABAT [4-aminobutyrate aminotransferase], ABCA1[ATP-binding cassette, sub-family A (ABC1), member 1], or ABCA13[ATP-binding cassette, sub-family A (ABC1), member 13], for example.Further examples of preferred conditions treatable with the presentsystem include may be selected from: Aicardi-Goutières Syndrome;Alexander Disease; Allan-Herndon-Dudley Syndrome: POLG-RelatedDisorders; Alpha-Mannosidosis (Type II and III); Alstrom Syndrome;Angelman; Syndrome; Ataxia-Telangiectasia; NeuronalCeroid-Lipofuscinoses; Beta-Thalassemia; Bilateral Optic Atrophy and(Infantile) Optic Atrophy Type 1; Retinoblastoma (bilateral); CanavanDisease; Cerebrooculofacioskeletal Syndrome 1 [COFS1]; CerebrotendinousXanthomatosis; Cornelia de Lange Syndrome; MAPT-Related Disorders;Genetic Prion Diseases; Dravet Syndrome; Early-Onset Familial AlzheimerDisease; Friedreich Ataxia [FRDA]; Fryns Syndrome; Fucosidosis: FukuyamaCongenital Muscular Dystrophy; Galactosialidosis; Gaucher Disease;Organic Acidemias; Hemophagocytic Lymphohistiocytosis;Hutchinson-Gilford Progeria Syndrome; Mucolipidosis II; Infantile FreeSialic Acid Storage Disease; PLA2G6-Associated Neurodegeneration;Jervell and Lange-Nielsen Syndrome; Junctional Epidermolysis Bullosa;Huntington Disease; Krabbe Disease (Infantile); MitochondrialDNA-Associated Leigh Syndrome and NARP; Lesch-Nyhan Syndrome;LIS1-Associated Lissencephaly; Lowe Syndrome; Maple Syrup Urine Disease;MECP2 Duplication Syndrome; ATP7A-Related Copper Transport Disorders;LAMA2-Related Muscular Dystrophy; Arylsulfatase A Deficiency;Mucopolysaccharidosis Types I, II or III; Peroxisome BiogenesisDisorders, Zellweger Syndrome Spectrum; Neurodegeneration with BrainIron Accumulation Disorders; Acid Sphingomyelinase Deficiency;Niemann-Pick Disease Type C; Glycine Encephalopathy; ARX-RelatedDisorders; Urea Cycle Disorders; COL1A1/2-Related OsteogenesisImperfecta; Mitochondrial DNA Deletion Syndromes; PLP1-RelatedDisorders; Perry Syndrome; Phelan-McDermid Syndrome; Glycogen StorageDisease Type II (Pompe Disease) (Infantile); MAPT-Related Disorders;MECP2-Related Disorders; Rhizomelic Chondrodysplasia Punctata Type 1;Roberts Syndrome; Sandhoff Disease; Schindler Disease—Type 1; AdenosineDeaminase Deficiency; Smith-Lemli-Opitz Syndrome; Spinal MuscularAtrophy; Infantile-Onset Spinocerebellar Ataxia; Hexosaminidase ADeficiency; Thanatophoric Dysplasia Type 1; Collagen Type VI-RelatedDisorders; Usher Syndrome Type I; Congenital Muscular Dystrophy;Wolf-Hirschhorn Syndrome; Lysosomal Acid Lipase Deficiency; andXeroderma Pigmentosum.

As will be apparent, it is envisaged that the present system can be usedto target any polynucleotide sequence of interest. Some examples ofconditions or diseases that might be usefully treated using the presentsystem are included in the Tables herein and examples of genes currentlyassociated with those conditions are also provided there. However, thegenes exemplified are not exhaustive.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: In Vivo Interrogation of Gene Function in the Mammalian BrainUsing CRISPR-Cas9

The publication by Swiech, L. et al. entitled “In vivo interrogation ofgene function in the mammalian brain using CRISPR-Cas9.” Nat Biotechnol.2014 Oct. 19. doi: 10.1038/nbt.3055. [Epub ahead of print] isincorporated herein by reference. This work presents the following mainpoints:

-   -   First demonstration of successful AAV-mediated Cas9 delivery in        vivo as well as efficient genome modification in post-mitotic        neurons;    -   Development of a nuclear tagging technique which enables easy        isolation of neuronal nuclei from Cas9 and sgRNA-expressing        cells;    -   Demonstration of application toward RNAseq analysis of neuronal        transcriptome;    -   Integration of electrophysiological studies with Cas9-mediated        genome perturbation; and    -   And demonstration of multiplex targeting and the ability to        study gene function on rodent behavior using Cas9-mediated        genome editing.

Transgenic animal models carrying disease-associated mutations areenormously useful for the study of neurological disorders, helping toelucidate the genetic and pathophysiological mechanism of disease.However, generation of animal models that carry single or multiplegenetic modifications is particularly labor intensive and requirestime-consuming breeding over many generations. Therefore, to facilitatethe rapid dissection of gene function in normal and disease-relatedbrain processes Applicants need ability to precisely and efficientlymanipulate the genome of neurons in vivo. The CRISPR-associatedendonuclease Cas9 from Streptococcus pyogenes (SpCas9) has been shown tomediate precise and efficient genome cleavage of single and multiplegenes in replicating eukaryotic cells, resulting in frame shiftinginsertion/deletion (indel) mutations. Here, Applicants integrateCas9-mediated genome perturbation with biochemical, sequencing,electrophysiological, and behavioral readouts to study the function ofindividual as wells as groups of genes in neural processes and theirroles in brain disorders in vivo.

Discussion

Adeno-associated viral (AAV) vectors are commonly used to deliverrecombinant genes into the mouse brain. The main limitation of the AAVsystem is its small packaging size, capped at approximately 4.5 kbwithout ITRs, which limits the amount of genetic material that can bepackaged into a single vector. Since the size of the SpCas9 is already4.2 kb, leaving less than 0.3 kb for other genetic elements within asingle AAV vector, Applicants designed a dual-vector system thatpackages SpCas9 (AAV-SpCas9) and sgRNA expression cassettes(AAV-SpGuide) on two separate viral vectors (FIG. 1). While designingthe AAV-SpCas9 vector, Applicants compared various short neuron-specificpromoters as well as poly adenylation signals to optimize SpCas9expression. For Applicants' final design Applicants chose the mouseMecp2 promoter (235 bp, pMecp2) and a minimal polyadenylation signal (48bp, spA) based on their ability to achieve sufficient levels of SpCas9expression in cultured primary mouse cortical neurons (FIG. 5c ). Tofacilitate immunofluorescence identification of SpCas9-expressingneurons, Applicants tagged SpCas9 with a HA-epitope tag. For theAAV-SpGuide vector, Applicants packaged an U6-sgRNA expression cassetteas well as the green fluorescent protein (GFP)-fused with the KASHnuclear trans-membrane domain9 driven by the human Synapsin I promoter(FIG. 1a ). The GFP-KASH fusion protein directs GFP to the outer nuclearmembrane (FIG. 5c,d ) and enables fluorescence-based identification andpurification of intact neuronal nuclei transduced by AAV-SpGuide.

To test the delivery efficacy of Applicants' dual-vector deliverysystem, Applicants first transduced cultured primary mouse corticalneurons in vitro and observed robust expression by AAV-SpCas9 andAAV-SpGuide (FIG. 5c ), with greater than 80% co-transduction efficiency(FIG. 5e ). Importantly, compared with un-transduced neurons, expressionof SpCas9 did not adversely affect the morphology and survival rate oftransduced neurons (FIG. 5c,f ).

Having established an efficient delivery system, Applicants next soughtto test SpCas9-mediated genome editing in mouse primary neurons. WhereasSpCas9 has been used to achieve efficient genome modifications in avariety of dividing cell types, it is unclear whether SpCas9 can be usedto efficiently achieve genome editing in post-mitotic neurons. ForApplicants' initial test Applicants targeted the Mecp2 gene, which playsa principal role in Rett syndrome, a type of autism spectrum disorder.MeCP2 protein is ubiquitously expressed in neurons throughout the brainbut nearly absent in glial cells and its deficiency has been shown to beassociated with severe morphological and electrophysiological phenotypesin neurons, and both are believed to contribute to the neurologicalsymptoms observed in patients with Rett syndrome. To target Mecp2,Applicants first designed several sgRNAs targeting exon 3 of the mouseMecp2 gene (FIG. 6a ) and evaluated their efficacy using Neuro-2a cells.The most efficient sgRNA was identified using the SURVEYOR nucleaseassay (FIG. 6b ). Applicants chose the most effective sgRNA (Mecp2target 5) for subsequent in vitro and in vivo Mecp2 targetingexperiments.

To assess the editing efficiency of Applicants' dual-vector system inneurons, Applicants transduced primary mouse cortical neurons at 7 daysin vitro (7 DIV, FIG. 7a ) and measured indel rate using the SURVEYORnuclease assay 7 days post transduction (FIG. 7b ). Of note, neuronculture co-transduced with AAV-SpCas9 and AAV-SpGuide targeting Mecp2showed up to 80% reduction in MeCP2 protein levels compared to controlneurons (FIG. 7c,d ). One possible explanation for the observeddiscrepancy between relatively low indel frequency (˜14%) and robustprotein depletion (˜80%) could be that mere binding by SpCas9 at thetarget site may interfere with transcription, which has been shown in E.coli. Applicants investigated this possibility using a mutant of SpCas9with both RuvC and HNH catalytic domains inactivated (D10A and H840A,dSpCas9). Co-expression of dSpCas9 and Mecp2-targeting sgRNA did notreduce MeCP2 protein levels (FIG. 7a,d ), suggesting that the observeddecrease of MeCP2 level in presence of active SpCas9 is due tooccurrence of modification in the Mecp2 locus. Another possibleexplanation for the discrepancy between the low level of detected indeland high level of protein depletion may be due to underestimation of thetrue indel rate by the SURVEYOR nuclease assay—the detection accuracy ofSURVEYOR has been previously shown to be sensitive to the indel sequencecomposition

MeCP2 loss-of-function has been previously shown to be associated withdendritic tree abnormalities and spine morphogenesis defects in neurons.These phenotypes of MeCP2 deprivation have also been reproduced inneurons differentiated from MeCP-KO iPS cells. Therefore, Applicantsinvestigated whether SpCas9-mediated MeCP2-depletion in neurons cansimilarly recapitulate morphological phenotypes of Rett syndrome.Indeed, neurons co-expressing SpCas9 and Mecp2-targeting sgRNA exhibitedaltered dendritic tree morphology and spine density when compared withcontrol neurons (FIG. 8). These results demonstrate that SpCas9 can beused to facilitate the study of gene functions in cellular assays byenabling targeted knockout in post-mitotic neurons.

Given the complexity of the nervous system, which consists of intricatenetworks of heterogeneous cell types, being able to efficiently edit thegenome of neurons in vivo would enable direct testing of gene functionin relevant cell types embedded in native contexts. Consequently,Applicants stereotactically injected a mixture (1:1 ratio) of high titerAAV-SpCas 9 and AAV-SpGuide into the hippocampal dentate gyrus in adultmice. Applicants observed high co-transduction efficiency of bothvectors (over 80%) in hippocampal granule cells at 4 weeks after viralinjection (FIG. 1b,c ) resulting in genomic modifications of the Mecp2locus. (FIG. 1d ). Using SURVEYOR nuclease assay Applicants detected˜13% indel frequency in brain punches obtained from injected brainregions (FIG. 1e ). Similar to Applicants' finding in cultured primaryneurons, SpCas9-mediated cutting of the Mecp2 locus efficientlydecreased MeCP2 protein levels by over 60% (FIG. 1f ). Additionally thenumber of MeCP2-positive nuclei in the dentate gyrus decreased by over75% when injected with AAV-SpCas 9 and AAV-SpGuide compared toAAV-SpCas9 alone (FIG. 1g-h ). These results suggest that SpCas9 can beused to directly perturb specific genes within intact biologicalcontexts.

Targeted genomic perturbations can be coupled with quantitative readoutsto provide insights into the biological function of specific genomicelements. To facilitate analysis of AAV-SpCas9 and AAV-SpGuidetransduced cells, Applicants developed a method to purify GFP-KASHlabeled nuclei using fluorescent activated cell sorting (FACS) (FIG. 2a). Sorted nuclei can be directly used to purify nuclear DNA and RNA fordownstream biochemical or sequencing analysis. Using sanger sequencing,Applicants found that 13 out of 14 single GFP-positive nuclei containedan indel mutation at the sgRNA target site.

In addition to genomic DNA sequencing, purified GFP-positive nuclei canalso be used for RNAseq analysis to study transcriptional consequencesof MeCP2 depletion (FIG. 2b and FIG. 9). To test the effect of Mecp2knockout on transcription of neurons from the dentate gyrus, Applicantsprepared RNAseq libraries using FACS purified GFP⁺ nuclei from animalsreceiving AAV-SpCas9 as well as either a control sgRNA that has beendesigned to target bacterial lacZ gene and not the mouse genome, or aMecp2-targeting sgRNA. All sgRNAs have been optimized to minimize theiroff-target score (CRISPR Design Tool). Applicants were able to finddifferentially expressed genes (FIG. 2b ) between control and Mecp2sgRNA expressing nuclei (p<0.01). Applicants identified severalinteresting candidates among genes that were down-regulated in Mecp2sgRNA expressing nuclei: Hpca, Olfm1, and Ncdn, which have beenpreviously reported to play important roles in learning behaviors; andCplx2, which has been shown to be involved in synaptic vesicle releaseand related to neuronal firing rate. These results demonstrate that thecombination of SpCas9-mediated genome perturbation and population levelRNAseq analysis provides a way to characterize transcriptionalregulations in neurons and suggest genes that may be important tospecific neuronal functions or disease processes.

SpCas9-mediated in vivo genome editing in the brain can also be coupledwith electrophysiological recording to study the effect of genomicperturbation on specific cell types or circuit components. To study thefunctional effect of MeCP2 depletion on neuronal physiology Applicantsstereotactically co-delivered AAV-SpCas9 and AAV-SpGuide targeting Mecp2into the superficial layer of the primary visual cortex (V1) of malemice. V1 was chosen since the superficial layer cortical excitatoryneurons are more accessible to two-photon imaging and two-photon guidedtargeted recording. Two weeks after SpCas9 delivery, mice were subjectedto two-photon guided juxtacellular recordings (FIG. 3) to compare theelectrophysiological response of KASH-GFP⁺ neurons and GFP⁻ neighboringneurons in layer 2/3 of mouse V1 (FIG. 3a-c ). Applicants measuredneuronal responses to 18 drifting gratings in 20-degree increments andcalculated evoked firing rate (FR) and orientation selectivity index(OSI) of cells by vector averaging the response. Both FR and OSI weresignificantly reduced for excitatory GFP⁺, MeCP2 knockout neurons,compared to neighboring GFP⁻ excitatory neurons (FIG. 3d-e ). Incomparison, control sgRNA expression together with SpCas9 did not haveany effect on FR and OSI when compared with neighboring uninfectedneurons (FIG. 3d-e ). These results show that SpCas9 mediated depletionof MCCP2 in adult V1 cortical neurons alters the visual responseproperties of excitatory neurons in vivo within two weeks and furtherdemonstrate the versatility of SpCas9 in facilitating targeted geneknockout in the mammalian brain in vivo, for studying genes functionsand dissection of neuronal circuits.

One key advantage of the SpCas9 system is its ability to facilitatemultiplex genome editing. Introducing stable knockouts of multiple genesin the brain of living animals will have potentially far-reachingapplications, such as causal interrogation of multigenic mechanisms inphysiological and neuropathological conditions. To test the possibilityof multiplex genome editing in the brain Applicants designed a multiplexsgRNA expression vector consisting of three sgRNAs in tandem, along withGFP-KASH for nuclei labeling (FIG. 4a ). Applicants chose sgRNAstargeting the DNA methyltransferases gene family (DNMTs), which consistsof Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 and 3a are highly expressed in theadult brain and it was previously shown that DNMT activity alters DNAmethylation and both Dnmt3a and Dnmt1 are required for synapticplasticity and learning and memory formation. Applicants designedindividual sgRNAs against Dnmt3a and Dnmt1 with high modificationefficiency. To avoid any potential compensatory effects by Dnmt3bApplicants decided also to additionally target this gene even though itis expressed mainly during neurodevelopment²⁷. Applicants finallyselected individual sgRNAs for high simultaneous DNA cleavage for allthree targeted genes (FIG. 4b and FIG. 10).

To test the efficacy of multiplex genome editing in vivo, Applicantsstereotactically delivered a mixture of high titer AAV-SpCas9 andAAV-SpGuide into the dorsal and ventral dentate gyrus of male adultmice. After 4 weeks, hippocampi were dissected and targeted cell nucleiwere sorted via FACS. Applicants detected ˜19% (Dnmt3a), 18% (Dnmt1) and4% (Dnmt3b) indel frequency in the sorted nuclei population usingSURVEYOR nuclease assay (FIG. 4c ) and sequencing (FIG. 11). Targetingmultiple loci raises the question about the effective rate ofmultiple-knockouts in individual cells. By using single nuclei sortingcombined with targeted sequencing, Applicants quantified simultaneoustargeting of multiple DNMT loci in individual neuronal nuclei (FIG. 4d). Of neuronal nuclei carrying modification in at least one Dnmt locus,more than 70% of nuclei contained indels in both Dnmt3a and Dnmt1 (˜40%contained indels at all 3 loci, and ˜30% at both Dnmt3a and Dnmt1 loci).These results are in agreement with Dnmt3a and Dnmt1 protein depletionlevels in the dentate gyrus (FIG. 4e ). Due to the low expression ofDnmt3b in the adult brain, Applicants were not able to detect Dnmt3bprotein.

Recent studies with SpCas9 have shown that, although each base withinthe 20-nt sgRNA sequence contributes to overall specificity, genomicloci that partially match the sgRNA can result in off-target doublestrand brakes and indel formations. To assess the rate of off-targetmodifications, Applicants computationally identified a list of highlysimilar genomic target sites² and quantified the rate of modificationsusing targeted deep sequencing. Indel analysis of the top predictedoff-target loci revealed a 0-1.6% rate of indel formations demonstratingthat SpCas9 modification is specific (Supplementary Table 1). Toincrease the specificity of SpCas9-mediated genome editing in vivo,future studies may use off-targeting minimization strategies such asdouble nicking and truncated sgRNAs.

Knockdown of Dnmt3a and Dnmt1 have been previous shown to impacthippocampus-dependent memory formation²⁷. Consequently, Applicantsperformed contextual fear-conditioning behavior tests to investigate theeffect of SpCas9-mediated triple knockout (Dnmt3a, Dnmt1 and Dnmt3b) onmemory acquisition and consolidation. While Applicants did not observeany differences between control and triple knockout mice in the memoryacquisition phase, knockout mice showed impaired memory consolidationwhen tested under trained context conditions (FIG. 4f ). This effect wasabolished when mice were tested in the altered context. Applicants'results demonstrate that CRIPSR-Cas9-mediated knockout of DNMT familymembers in dentate gyrus neurons is sufficient to probe the function ofgenes in behavioral tasks.

Applicants' results demonstrate that AAV-mediated in vivo delivery ofSpCas9 and sgRNA provides a rapid and powerful technology for achievingprecise genomic perturbations within intact neural circuits. WhereasSpCas9 has been broadly used to engineer dividing cells, Applicantsdemonstrate that SpCas9 can also be used to engineer the genome ofpostmitotic neurons with high efficiency via NHEJ-mediated indelgeneration. SpCas9-mediated genomic perturbations can be combined withbiochemical, sequencing, electrophysiological, and behavioral analysisto study the function of the targeted genomic element. Applicantsdemonstrated that SpCas9-mediated targeting of single or multiple genescan recapitulate morphological, electrophysiological, and behavioralphenotypes observed using classical, more time-consuming genetic mousemodels. The current study employed the Streptococcus pyogenes Cas9,which not only necessitates the use of two AAV vectors but also limitsthe size of promoter elements can be used to achieve cell type-specifictargeting. Given the diversity of Cas9 orthologues, with some beingsubstantially shorter than SpCas9, it should be possible to engineersingle AAV vectors expressing both Cas9 and sgRNA, as described herein.

Methods

DNA Constructs

For SpCas9 targets selection and generation of single guide RNA (sgRNA),the 20-nt target sequences were selected to precede a 5′-NGG PAMsequence. To minimize off-targeting effects, the CRIPSR design tool wasused. sgRNA was PCR amplified using U6 promoter as a template withforward primer: 5′-cgcacgcgtaattcgaacgctgacgtcatc-3′ (SEQ ID NO: 43) andreverse primer containing the sgRNA with 20-nt DNA target site (Bold):5′-cacacgcgtAAAAAAgcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctaTTTCtagctctaaaacNNNNNNNNNNNNNNNNNNCGGTGTTTCGTCCTTTCCAC-3′. (SEQ ID NO: 44) Control sgRNA sequence was designed totarget lacZ gene from E. coli:

target sequence:  (SEQ ID NO: 45) TGCGAATACGCCCACGCGATGGG

EGFP-KASH construct was a generous gift from Prof. Worman (ColumbiaUniversity, NYC) and was used as PCR template for cloning the codingcassette into AAV backbone under the human Synapsin promoter (hSyn).Next, U6-Mecp2sgRNA coding sequence was introduced using MluI site. Forthe multiplex gene targeting strategy, individual sgRNAs were PCRamplified as described above. All three sgRNAs were ligated with PCRamplified hSyn-GFP-KASH-bGHpA cassette (see Figure. 1A) by using theGolden Gate cloning strategy. After PCR amplification, the Golden Gateligation product containing 3 sgRNAs and hSyn-GFP-KASH-bGH pA was clonedinto AAV backbone. All obtained constructs were sequenced verified. Inorder to find the optimal promoter sequence to drive SpCas9 expressionin neurons Applicants tested: hSyn1, mouse truncated Mecp2 (pMecp2), andtruncated rat Map1b (pMap1b) promoter sequences² (see FIG. 5a ).Following primers were used to amplify promoter regions:

hSyn_F:  (SEQ ID NO: 46) 5′-GTGTCTAGACTGCAGAGGGCCCTG-3′; hSyn_R: (SEQ ID NO: 47) 5′-GTGTCGTGCCTGAGAGCGCAGTCGAGAA-3′; Mecp2_F (SEQ ID NO: 48) 5′-gagaagcttAGCTGAATGGGGTCCGCCTC-3′; Mecp2_R (SEQ ID NO: 49) 5′-ctcaccggtGCGCGCAACCGATGCCGGGACC-3′; Map1b-283/-58_F (SEQ ID NO: 50) 5′-gagaagcttGGCGAAATGATTTGCTGCAGATG-3′; Map1b-283/-58_R (SEQ ID NO: 51) 5′-ctcaccggtGCGCGCGTCGCCTCCCCCTCCGC-3′.

Another truncation of rat map1b promoter was assembled with thefollowing oligos:

(SEQ ID NO: 52) 5′-agcttCGCGCCGGGAGGAGGGGGGACGCAGTGGGCGGAGCGGAGACAGCACCTTCGGAGATAATCCTTTCTCCTGCCGCAGAGCAGAGGAGCGGCGGGAGAGGAACACTTCTCCCAGGCTTTAGCAGAGCCGGa-3′ and (SEQ ID NO: 53)5′-ccggtCCGGCTCTGCTAAAGCCTGGGAGAAGTGTTCCTCTCCCGCCGCTCCTCTGCTCTGCGGCAGGAGAAAGGATTATCTCCGAAGGTGCTGTCTCCGCTCCGCCCACTGCGTCCCCCCTCCTCCCGGCGCGa-3′.

Short synthetic polyadenylation signal (spA)³ was assembled usingfollowing oligos:

(SEQ ID NO: 54) 5′-aattcAATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTG TGTgc-3′ and (SEQ ID NO: 55)5′-ggccgcACACAAAAAACCAACACACAGATCTAATGAAAATAAAGAT  CTTTTATTg-3′. 

SpCas9 and its D10A mutant version (dSpCas9) were describedpreviously^(4, 5). Plasmid encoding red fluorescent protein (mCherry)under control of EFIa promoter was used for neuron transfection withLipofectamine® 2000 (Life Technologies).

Cell Line Cultures and Transfection

Neuro-2a (N2a) cells were grown in DMEM containing 5% fetal bovine serum(BSA). For HEK293FT cells DMEM containing 10% fetal bovine serum (FBS)was used. Cells were maintained at 37° C. in 5% CO₂ atmosphere. Cellswere transfected using Lipofectamine® 2000 or Polyethylenimine (PEI)“MAX” reagent (Polysciences), according to manufacturer's protocols.

Production of Concentrated AAV Vectors

High titer AAV1/2 particles were produced using AAV1 and AAV2 serotypeplasmids at equal ratios and pDF6 helper plasmid and purified on heparinaffinity column⁶. Titering of viral particles was done by qPCR. Hightiter AAV1 particles were produced by the UNC Vector Core Services(University of North Carolina at Chapel Hill). Low titer AAV1 particlesin DMEM were produced as described previously⁷. Briefly, HEK293FT cellswere transfected with transgene plasmid, pAAV1 serotype plasmid and pDF6helper plasmid using PEI “MAX”. Culture medium was collected after 48 hand filtered through a 0.45 μm PVDF filter (Millipore).

Primary Cortical Neuron Culture

Animals used to obtain neurons for tissue cultures were sacrificedaccording to the protocol approved by the MIT Committee on Animal Care(MIT CAC). Primary cultures were prepared from embryonic day 16 mousebrains⁸. Embryos of either sex were used. Cells were plated onpoly-D-lysine (PDL) coated 24-well plates (BD Biosciences) orlaminin/PDL coated coverslips (VWR). Cultures were grown at 37° C. and5% CO₂ in Neurobasal medium, supplemented with B27, Glutamax (LifeTechnologies) and penicillin/streptomycin mix. For AAV transduction,cortical neurons in 500 μl Neurobasal culture medium were incubated at 7DIV with 300 μl (double infection at 1:1 ratio) AAV1-containingconditioned medium from HEK293FT cells⁷. One week after transductionneurons have been harvested for downstream processing or fixed in 4%paraformaldehyde for immunofluorescent stainings or morphology analysis.

For visualization of neuronal morphology, cells at DIV7 were transfectedwith EF1α-mCherry expression vector using Lipofectamine® 2000 (LifeTechnologies) for one week as previously described⁹. For measurement oftotal dendrite length, all dendrites of individual neurons were tracedusing ImageJ software. Quantification of the number of primarydendrites, dendritic tips and the Sholl analysis¹⁰ were performed onimages acquired with fluorescent microscope at a 40× objective (ZeissAxioCam Ax10 microscope, Axiocam MRm camera). For dendrites number, endsof all non-axonal protrusions longer than 10 μm were counted. For Shollanalysis, concentric circles with 5 μm step in diameter wereautomatically drawn around the cell body, and the number of dendritescrossing each circle was counted using ImageJ software with a Shollplug-in.

Stereotactic Injection of a AV1/2 into the Mouse Brain

The MIT CAC approved all animal procedures described here. Adult (12-16weeks old) male C57BL/6N mice were anaesthetized by intraperitoneal(i.p.) injection of 100 mg/kg Ketamine and 10 mg/kg Xylazine.Pre-emptive analgesia was given (Buprenex, 1 mg/kg, i.p.). Craniotomywas performed according to approved procedures and 1 μl of 1:1 AAVmixture (1×1013 Vg/ml of sMecp2-SpCas9; 6×1012 Vg/ml of DNMT 3×sgRNA;3-5×1012 Vg/ml of hSyn-GFP-KASH) was injected into: dorsal dentate gyrus(anterior/posterior: −1.7; mediolateral: 0.6; dorsal/ventral: −2.15)and/or ventral dentate gyrus (anterior/posterior: −3.52; mediolateral:2.65; dorsal/ventral: −3). For in vivo electrophysiology recordingsexperiments (FIG. 3) virus injection coordinates were 3 mm lateral (fromBregma) and 1 mm anterior from the posterior suture. The skull wasthinned using a dremel drill with occasional cooling with saline, andthe remaining dura was punctured using a glass micropipette filled withthe virus suspended in mineral oil. Several injections (3-4) were madeat neighboring sites, at a depth of 200-250 μm. A volume of 150-200 nlof virus mixture was injected at 75 nl/min rate at each site. After eachinjection, the pipette was held in place for 3-5 minutes prior toretraction to prevent leakage. The incision was sutured and properpost-operative analgesics (Meloxicam, 1-2 mg/kg) were administered forthree days following surgery.

In Vivo Two-Photon Guided Targeted Loose Patch Recordings

Two weeks after virus injection, mice were used for electrophysiologyexperiments. Mice were anesthetized with 2% isoflurane and maintainedusing 0.8% isoflurane. The skin was excised, cleaned with sugi and ametal head plate was attached to the skull using glue and dentalacrylic, and a 2 mm×2 mm craniotomy was performed over the primaryvisual cortex (V1). The exposed area was then covered with a thin layerof 1.5% agarose in artificial cerebrospinal fluid (aCSF; 140 mM NaCl, 5mM KCl, 2 mM CaCl₂), 1 mM MgCl2, 0.01 mM EDTA, 10 mM HEPES, 10 mMglucose; pH 7.4). Animal body temperature was maintained duringexperiment 37.5° C. with a heating blanket. Borosilicate pipettes (WPI)were pulled using a Sutter P-2000 laser puller (Sutter Instruments). Tipdiameter was around 1 μm while the resistance was between 3-5 MΩ.Recordings were made using custom software (Network Prism, Sur lab),written in Matlab (MathWorks), controlling a MultiClamp 700B amplifier(Axon). A glass pipette electrode was inserted into the brain at anangle of 20-35° and an Ag/AgCl ground electrode pellet (WarnerInstruments) was positioned in the same solution as the brain and theobjective. For fluorescent visualization, pipettes were filled withAlexa Fluor 594 (Molecular Probes). The pipette was first targeted tothe injection site using a 10× lens, and then targeted to individualGFP+ cells using a 25× lens via simultaneous two-photon imaging at 770nm. Cell proximity was detected through deflections in resistanceobserved in voltage clamp during a rapidly time-varying 5 mV commandvoltage pulse. Once resistance had increased by 5-10 MΩ, the amplifierwas switched to current clamp, and spikes were recorded with zeroinjected current, under a Bessel filter of 4 KHz and an AC filter of 300Hz. Virus injected brains were perfused post hoc andimmunohistochemistry was performed.

Visual Stimulation and Data Analysis from In Vivo Two-Photon GuidedTargeted Loose Patch Recordings

To assess the orientation selectivity and tuning of genome-editedneurons, Applicants presented oriented gratings using custom softwarewritten in Matlab PsychToolbox-3. Gratings were optimized for cellularresponsiveness and were presented by stepping the orientation from 0-360degrees in steps of 20 degrees, with each grating presentation beingpreceded for 4 seconds “off” followed by 4 seconds “on”, for a totalpresentation duration of 144 seconds.

Data was acquired directly into Matlab and saved as .mat files. Spikedetection was performed via analysis routines that used manually definedthresholds followed by spike shape template matching for furtherverification. Every spike was tagged and displayed on screen in agraphical user interface whereupon it was manually reviewed for falsepositives and negatives by the experimenter. Spike times in response toevery stimulus were then grouped into “on” or “off” periods based ontheir timing relative to visual stimulation, and “on” spikes for eachstimulus were decremented by the number of “off” spikes observed duringan equal time period. For orientation experiments, # spikes perstimulus=(# spikes “on”)−(# spikes “off”) because “on” and “off” periodswere the same duration. For every cell of interest, the methods wereused to collect responses for each oriented stimulus (0 to 360 degrees,in steps of 20 degrees). These responses were then turned into a “tuningcurve” of orientation vs. response for each trial. OrientationSelectivity Index (OSI) was computed by taking the vector average forthe preferred orientation according to the formulae as follows:

${OSI} = \frac{\sqrt{\left( {\sum\limits_{i}{{R\left( \theta_{i} \right)}{\sin\left( {2\theta_{i}} \right)}}} \right)^{2} + \left( {\sum\limits_{i}{{R\left( \theta_{i} \right)}{\cos\left( {2\theta_{i}} \right)}}} \right)^{2}}}{\sum\limits_{i}{R\left( \theta_{i} \right)}}$

Tissue Preparation and Purification of Cell Nuclei

Total hippocampus or dentate gyrus was quickly dissected in ice coldDPBS (Life Sciences) and shock frozen on dry ice. For cell nucleipurification, tissue was gently homogenized in 2 ml ice-coldhomogenization buffer (HB) (320 mM Sucrose, 5 mM CaCl, 3 mM Mg(Ac)₂, 10mM Tris pH7.8, 0.1 mM EDTA, 0.1% NP40, 0.1 mM PMSF, 1 mMbeta-mercaptoethanol) using 2 ml Dounce homogenizer (Sigma); 25 timeswith pestle A, followed by 25 times with pestle B. Next, 3 ml of HB wasadded up to 5 ml total and kept on ice for 5 min. For gradientcentrifugation, 5 ml of 50% OptiPrep™ density gradient medium (Sigma)containing 5 mM CaCl, 3 mM Mg(Ac)₂, 10 mM Tris pH 7.8, 0.1 mM PMSF, 1 mMbeta-mercaptoethanol was added and mixed. The lysate was gently loadedon the top of 10 ml 29% iso-osmolar OptiPrep™ solution in a conical 30ml centrifuge tube (Beckman Coulter, SW28 rotor). Samples werecentrifuged at 10,100×g (7,500 rpm) for 30 min at 4° C. The supernatantwas removed and the nuclei pellet was gently resuspended in 65 mMbeta-glycerophosphate (pH 7.0), 2 mM MgCl₂, 25 mM KCl, 340 mM sucroseand 5% glycerol. Number and quality of purified nuclei was controlledusing bright field microscopy.

Cell Nuclei Sorting

Purified GFP-positive (GFP⁺) and negative (GFP⁻) intact nuclei wereco-labeled with Vybrant® DyeCycle™ Ruby Stain (1:500, Life Technologies)and sorted using BD FACSAria III (Koch Institute Flow Cytometry Core,MIT). GFP⁺ and GFP⁻ nuclei were collected in 1.5 ml Eppendorf tubescoated with 1% BSA and containing 400 μl of resuspension buffer (65 mMbeta-glycerophosphate pH 7.0, 2 mM MgCl₂, 25 mM KCl, 340 mM sucrose and5% glycerol). After sorting, all samples were kept on ice andcentrifuged at 10,000×g for 20 min at 4° C. Nuclei pellets were storedat −80° C. or were directly used for downstream processing.

Genomic DNA Extraction and SURVEYOR™ Assay

For functional testing of sgRNA, 50-70% confluent N2a cells wereco-transfected with a single PCR amplified sgRNA and SpCas9 vector.Cells transfected with SpCas9 only served as negative control. Cellswere harvested 48 h after transfection, and DNA was extracted usingDNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer'sprotocol. To isolate genomic DNA from AAV1 transduced primary neurons,DNeasy Blood & Tissue Kit was used 7 days post AAV transduction,according to the manufacturer's instruction. Sorted nuclei or dissectedtissues were lysed in lysis buffer (10 mM Tris, pH 8.0, 10 mM NaCl, 10mM EDTA, 0.5 mM SDS, Proteinase K (PK, 1 mg/ml) and RNAse A) at 55° C.for 30 min. Next, chloroform-phenol extraction was performed followed byDNA precipitation with ethanol, according to standard procedures. DNAwas finally resuspended in TE Buffer (10 mM Tris pH 8.0, 0.1 mM EDTA)and used for downstream analysis. Functional testing of individualsgRNAs was performed by SURVEYOR™ nuclease assay (Transgenomics) usingPCR primers listed in Supplementary Table 2. Band intensityquantification was performed as described herein

RNA Library Preparation and Sequencing

Two weeks after bilateral viral delivery of SpCas9 with guide targetingMecp2 (4 animals) or SpCas9 with gRNA targeting lacZ (4 animals), thedentate gyrus was quickly dissected in ice cold DPBS (Life Sciences) andtransferred immediately to RNA-later solution (Ambion). After 24 hoursin 4° C. the tissue was moved to −80° C. Populations of 100 targetedneuronal nuclei were FACS sorted into 10 μl TCL buffer supplemented with1% 2-mercaptoethanol (Qiagen). After centrifuging, samples were frozenimmediately at −80° C. The RNA was purified by AMPure RNAcleanXP SPRIbeads (Beckman Coulter Genomics) following the manufactures'instructions, and washed three times with 80% ethanol, omitting thefinal elution. The beads with captured RNA were air-dried and processedimmediately for cDNA synthesis. Samples with no nuclei were used asnegative controls. Three population samples were used for each animal,total of 24 population sample, in cDNA library preparations followingthe SMART-seq2 protocol only replacing the reverse transcriptase enzymewith 0.1 ul of Maxima H Minus enzyme (200 U/ul, Thermo Scientific), andscaling down the PCR reaction to a volume of 25 ul. The tagmentationreaction and final PCR amplification were done using the Nextera XT DNASample preparation kit (Illumina), with the following modifications. Allreaction volumes were scaled down by a factor of 4, and the librarieswere pooled after the PCR amplification step by taking 2.5 ul of eachsample. The pooled libraries were cleaned and size-selected using tworounds of 0.7 volume of AMPure XP SPRI bead cleanup (Beckman CoulterGenomics). Samples were loaded on a High-Sensitivity DNA chip (Agilent)to check the quality of the library, while quantification was done withQubit High-Sensitivity DNA kit (Invitrogen). The pooled libraries werediluted to a final concentration of 4 nM and 12 pmol and were sequencedusing Illumina Miseq with 75 bp paired end reads.

RNA Libraries Data Analysis

Bowtie2 index was created based on the mouse mm9 UCSC genome and knownGene transcriptome¹³, and paired-end reads were aligned directly to thisindex using Bowtie2 with command line options -q-phred33-quals -n 2-e99999999-1 25-I 1-X 1000-a-m 200-p 4-chunkmbs 512. Next, RSEM v1.27 wasrun with default parameters on the alignments created by Bowtie2 toestimate expression levels. RSEM's gene level expression estimates (tau)were multiplied by 1,000,000 to obtain transcript per million (TPM)estimates for each gene, and TPM estimates were transformed to log-spaceby taking log 2(TPM+1). Genes were considered detected if theirtransformed expression level equal to or above 2 (in log 2(TPM+1)scale). A library is filtered out if it has less than 8000 genesdetected. Based on this criterion, 4 libraries were filtered andexcluded from the downstream analysis. To find differentially expressedgenes between control animals and Mecp2 sgRNA expressing animals,Student's t-test (Matlab V2013b) and cross validation was used in 20random permutation runs, where in each run one library from each animalwas randomly chosen to exclude (this results in a total of 12 librariesused in the t-test each time). The t-test was run on all genes that havemean expression level above 0.9 quantile (usually around 5 log 2(TPM+1))for each sample. Then, genes that were significant (p<0.01) in more thanone thirds of the permutation runs were chosen. The log 2(TPM+1)expression levels of these genes across samples were clustered usinghierarchical clustering (Matlab V2013b).

Immunofluorescent Staining

Cell culture: For immunofluorescent staining of primary neurons, cellswere fixed 7 days after viral delivery with 4% paraformaldehyd (PFA) for20 min at RT. After washing 3 times with PBS, cells were blocked with 5%normal goat serum (NGS) (Life Technologies), 5% donkey serum (DS)(Sigma) and 0.1% Triton-X100 (Sigma) in PBS for 30 min at RT. Cells wereincubated with primary antibodies in 2.5% NGS, 2.5% DS and 0.1%Triton-X100 for 1 hour at RT or overnight at 4° C. After washing 3 timeswith PBST, cells were incubated with secondary antibodies for 1 hour atRT. Finally, coverslips were mounted using VECTASHIELD HardSet MountingMedium with DAPI (Vector Laboratories) and imaged using an Zeiss AxioCamAx10 microscope and an Axiocam MRm camera. Images were processed usingthe Zen 2012 software (Zeiss). Quantifications were performed by usingImageJ software 1.48 h and Neuron detector plugin. Mice were sacrified 4weeks after viral delivery by a lethal dose of Ketamine/Xylazine andtranscardially perfused with PBS followed by PFA. Fixed tissue wassectioned using vibratome (Leica, VT1000S). Next, 30 μm sections wereboiled for 2 min in sodium citrate buffer (10 mM tri-sodium citratedehydrate, 0.05% Tween20, pH 6.0) and cool down at RT for 20 min.Sections were blocked with 4% normal goat serum (NGS) in TBST (137 mMNaCl, 20 mM Tris pH 7.6, 0.2% Tween-20) for 1 hour. Paraffin sectionswere cut using a microtom (Leica RM2125 RTS) to 8 μm, and stained asdescribed previously. Sections were incubated with primary antibodiesdiluted in TBST with 4% NGS overnight at 4° C. After 3 washes in TBST,samples were incubated with secondary antibodies. After washing withTBST 3 times, sections were mounted using VECTASHIELD HardSet MountingMedium with DAPI and visualized with confocal microscope (Zeiss LSM 710,Ax10 ImagerZ2, Zen 2012 Software). Following primary antibodies wereused: rabbit anti-Dnmt3a (Santa Cruz, 1:100); rabbit anti-MeCP2(Millipore, 1:200); mouse anti-NeuN (Millipore, 1:50-1:400); chickenanti-GFAP (Abcam, 1:400); mouse anti-Map2 (Sigma, 1:500); chickenanti-GFP (Aves labs, 1:200-1:400); mouse anti-HA (Cell Signaling,1:100). Secondary antibodies: AlexaFluor® 488, 568 or 633 (LifeTechnologies, 1:500-1:1,000).

Quantification of LIVE/DEAD® Assay

Control and transduced primary neurons were stained using the LIVE/DEAD®assay (Life technologies) according to the manufacturer's instruction.To avoid interference with the GFP-signal from GFP-KASH expression,cells were stained for DEAD (ethidium homodimer) and DAPI (all cells)only. Stained cells were imaged using fluorescence microscopy and DEAD,GFP and DAPI positive cells were counted by using ImageJ 1.48 h softwareand Neuron detector plugin.

Western Blot Analysis

Transduced primary cortical neurons (24 well, 7 days after viraldelivery) and transduced tissue samples (4 weeks after viral delivery)were lysed in 50 μL of ice-cold RIPA buffer (Cell Signaling) containing0.1% SDS and proteases inhibitors (Roche, Sigma). Cell lysates weresonicated for 5 min in a Bioruptor sonicater (Diagenode) and proteinconcentration was determined using the BCA Protein Assay Kit (PierceBiotechnology, Inc.). Protein lysats were dissolved in SDS-PAGE samplebuffer, separated under reducing conditions on 4-15% Tris-HCl gels(Bio-Rad) and analyzed by Western blotting using primary antibodies:rabbit anti-Dnmt 3a (Santa Cruz, 1:500), mouse anti-Dnmt1 (NovusBiologicals, 1:800), rabbit anti-Mecp2 (Millipore, 1:400), rabbitanti-Tubulin (Cell Signaling, 1:10,000) followed by secondary anti-mouseand anti-rabbbit HRP antibodies (Sigma-Aldrich, 1:10,000). GAPDH wasdirectly visualized with rabbit HRP coupled anti-GAPDH antibody (CellSignaling, 1:10,000). Tubulin or GAPDH served as loading control. Blotswere imaged with ChemiDoc™ MP system with ImageLab 4.1 software(BioRad), and quantified using ImageJ software 1.48h.

Delay Contextual Fear Conditioning (DCFC)

8 weeks after bilateral SpCas9/DNMT 3×sgRNA delivery into the dorsal andventral dentate gyrus of 12 weeks old C57BL/6N male mice, animals werehabituated to the experimentor and the behavior room for 7 days.SpCas9/GFP-KASH injected littermates served as controls. At day 1 ofDCFC, mouse cages were placed into an islolated anterroom to preventmice from auditory cues before and after testing. Indivdual mice wereplaced into the FC chamber (Med Associates Inc.) and a 12 minhabituation period was performed. After habituation the mice were placedback to their homecages. The next day (training day) individual micewere placed into the chamber and were allowed to habituate for 4 min.After another 20 sec (pre-tone) interval, the tone (auditory cue) at alevel of 85 dB, 2.8 kHz was presented for 20 sec followed by 18 secdelay interval before the foot-shock was presented (0.5 mA, 2 sec).After the foot-shock, 40 sec interval (post-tone/shock) preceded a nextidentical trial starting with the 20 sec pre-tone period. The trainingtrial was repeated 6 times before the mice were placed back to theirhomecages. At day 3 (testing day), mice were first placed in theconditioning context chamber for 3 min. Next, mice underwent 4×100 sectesting trials starting with a 20 sec interval followed by 20 sec toneand a 60 sec post-tone interval. Finally, mice were placed in an alteredcontext-conditioning chamber (flat floor vs. grid, tetrameric vs.heptameric chamber, vanillin scent) and the testing trial was repeated.Freezing behavior was recorded and analysis was performed blind off-linemanually and confirmed with Noldus EthoVision XT software (NoldusInformation Technology).

Deep Sequencing Analysis and Indel Detection

CRISPR Design Tool was used to find potential off-targets for DNMTfamily genes, targeted by CRISPR-SpCas9 in the brain. Targeted cellnuclei from dentate gyrus were FACS sorted 12 weeks after viral deliveryand genomic DNA was purified as described above. For each gene ofinterest, the genomic region flanking the CRISPR target site wasamplified by a fusion PCR method to attach the Illumina P5 adapters aswell as unique sample-specific barcodes to the target amplicons (for on-and off-target primers see Supplementary Table 3). Barcoded and purifiedDNA samples were quantified by Qubit 2.0 Fluorometer (Life Technologies)and pooled in an equimolar ratio. Sequencing libraries were thensequenced with the Illumina MiSeq Personal Sequencer (LifeTechnologies), with read length 300 bp. The MiSeq reads were analyzed asdescribed previously in¹⁵. Briefly, reads were filtered by Phred quality(Q score) and aligned using a Smith-Waterman algorithm to the genomicregion 50 nucleotides upstream and downstream of the target site. Indelswere estimated in the aligned region from 5 nucleotides upstream to 5nucleotides downstream of the target site (a total of 30 bp). Negativecontrols for each sample were used to estimate the inclusion orexclusion of indels as putative cutting events. Applicants computed amaximum-likelihood estimator (MLE) for the fraction of reads havingtarget-regions with true-indels, using the per-target-region-per-readerror rate from the data of the negative control sample. The MLE scoresand cutting rates for each target are listed in Supplementary Table 1.

Statistical Analysis

All experiments were performed with a minimum of two independentbiological replicates. Statistics were performed with Prism6 (GraphPad)using Student's two tailed t-test.

Supplementary Table 1. Off-Target Analysis for DNMTs Targeting

Supplementary Table 1 Off-target analysis for DNIVITs targeting SEQPotential off- MLE ID Gene GI target sequences (%) SEM NO: Dnmt1 Abca1NM_013454 GGAGCTGGAGCTGTTCACGTTGG 0.0000 0.00 56 Mctp1 NM_030174CGGGCAGCAGATGTTCGCGTAGG 0.0806 0.08 57 Exd2 NM_133798AGGGCTTGAGATGTTCGGGCTGG 0.0612 0.06 58 Pik3r6 NM_001004435CCGGCTGGGGCTGTCCTCGCTAG 0.0000 0.00 59 Sobp NM_175407CGGGGTGCAGCTGCTCACGCCAG 0.0000 0.00 60 Vac14 NMJ46216CTGGCGGGAGCTGGTCGCGTGAG 0.0083 0.00 61 Dnmt3a Efemp2 NM_021474TGAGCATGGGCCGCTGGCGGTGG 0.0050 0.01 62 Bmpr1b NM_001277217ATGGCATAGGCCGCTGACAGAGG 0.0117 0.01 63 Syce1 NM_001143765TTGGCATGGTGAGCTGGCGGGGG 0.0067 0.00 64 Atp8b3 NM_026094TGGGCAGGGGTCTCTGAGGGCAG 0.0067 0.01 65 Rdh11 NM_021557TTGGCATGGGTCTCTTACCAAGG 0.0017 0.00 66 Dnmt3b Hecw2 NM_001001883ACATGGTTCCAGTGGGTATGTAG 0.0000 0.00 67 Plekhg3 NM_153804GGAGGTGGGCAGCGGGTATGTAG 0.0954 0.01 68 Cdc25b NM_001111075AGAAGGTCCCCGCGGGCATGGAG 0.2421 0.12 69 Top1mt NM_028404GGAGGGAACCAGCCGGTATGGGG 0.0167 0.01 70 Sesn2 NM_144907AGAGAGTGGCAGTGGGTAAGCAG 0.0000 0.00 71 Ncan NM_007789AGAGGTGGCCAGCGGGCAGGAAG 0.0017 0.00 72 Nacad NM_001081652TGAGGGGGCCAGCTGGGATGCAG 1.6254 0.76 73Supplementary Table 2. PCR Primers Used in the SURVEYOR Assay

Supplementary Table 2 PCR primers used in the SURVEYOR assay SEQ SEQForward primer ID Reverse primer ID Gene sequence (5′-3′) NO:sequence (5′-3′) NO: Mecp2 GGTCTCATGTGTGGCACTCA 74 TGTCCAACCTTCAGGCAAGG75 Dnmt3a ATCCCTCCTCAGAGGGTCAGC 76 TACCTCATGCACAGCTAGCACC 77 Dnmt1TTCGGGCATAGCATGGTCTTCC 78 GTTCTATTTCAGAGGGCTGATCCC 79 Dnmt3bGTTCTGAGCCGCACAGTTTGG 80 GGATAAGAAGGGACAATACAGG 81Supplementary Table 3. Primers Used for On- and Off-Target Genomic LociAmplification

Supplementary Table 3 Primers used for on- and off-target genomic lociamplification Forward primer SEQ ID  Reverse primer SEQ ID Genesequence (5′-3′) NO: sequence (5′-3′) NO: Dnmt1 GCCGGGGTCTC  82CTACCGCCTGCGGA  83 GTTCAGAGCT CATGGT Dnmt3a CCTGTCTCTCTGT  84CCGTTTGCTGATGTAGTA  85 CCTAGGGCTCC GGGGTCC Dnmt3b CCCACAGGAAA  86CATCCTTCGTGTCT  87 CAATGAAGGGAGAC GAGGACTGGTC Abca1 CCCTGACACCAGC  88CTCTGGGTGAC  89 TGTTCAGCAC CACACACGATGC Mctp1 GAGCAGGCAGA  90GGAGAGCGTCC  91 GCCGAGCAAG GCCAGGAG Exd2 GGGTCTTGTTGTG  92 GAAGCTCTCTTAA 93 AGTAGGGTGTG CTACTGTTC Pik3r6 CCTGGAATACTAT  94 CAGGCCCTAGCAGCG  95TTCCACGCCG AGCAG Sobp GCAGCACACTCCA  96 GGAAGGGGCTTTCC  97 CCCTCACATTCCGAGC Vac14 CGGCGTCACG  98 GCTCCGACCCTGCT  99 TGACCTGAGTAAC CTCCCAEfemp2 GTGTCTGCCTC 100 CCTGTTCATCAGGCTC 101 GCTCTGCTGC GTAGCCC Bmpr1bCTATCTGAAATCC 102 CGATTGCTGGCTTGC 103 ACCACCTTAGACGC CTTGAG Syce1GCCTGAGGGGG 104 GGTTCGCGTCCGCC 105 CCAGAGGT CGCGTGAT Atp8b3 GGGACTCC 106GAGAGGTGGTC 107 CCGGGTGGTG CTGTCGCCTATG Rdh11 GACCCTGTGTTT 108CCCAGCAGGTCACA 109 CAAGTCTCTCTG GCTGACATC Hecw2 GGCCATCCAGTAC 110AGCACAGTATGTATTC 111 ATTCAATACG TATAAAATAATACGAC Plekhg3 GCAGAAGCCGT 112GTGGGAGGGGACAG 113 GACTCACAGCA AGACCATG Cdc25b CTTGTGCTTG 114CCTTACCTGTTCCTCT 115 TGATTCTGTCCTTACTGC TCCTTATCCAGC Top1mt CGAGAAGTC116 ATACCCAGTCCAC 117 GATGCAGACACTTCAA ATCCCTGCC Sesn2 GCTGAAGACTGGC 118CCTCTGCATCTCCCTCAGGAAGT 119 GAGCACAGCT ATT Ncan GACCTGAATGTTG 120GCCTCCTGTC 121 TGGCTGAGAGTCC CCCAGGTCCC Nacad CCCTCACGTTCC 122CACTAGGCTT 123 TGTCCAGCAA GGGCTGCCCTCT

Example 2: In Vivo Retina Disease Model (Mouse Model for RetinitisPigmentosa)

Applicants demonstrate the in vivo effectiveness of a genome engineeringapproach using Cas9 when packaged into AAV, and have successfully usedit to modify endogenous genome sequence in mammalian cells. Applicantsuse this system to demonstrate genetic engineering potential in neurons,which represents one key group of post-mitotic cells in human body.Applicants' studies underscore the therapeutic potential of this in vivogenome engineering of somatic tissue through the correction of mutationsassociated with human disease Retinitis Pigmentosa in the mouse modelthat bears mutation corresponding to the similar genetic defects foundin human patients. The mouse strain used for Applicants' studies isC57BL/6 strain B6.129S6(Cg)-Rhotm1.1Kpal/J. This mouse strain was chosenbecause these mice carry a nucleotide substitution at codon 23 in themouse rhodopsin (Rho) gene, CCC to CAC. This codon encodes the aminoacid substitution of histidine for phenylalanine at position 23, P23H.The P23H mutation is one of the most common causes of autosomal dominantretinitis pigmentosa. The genomic location of the Rho gene is on mousechromosome 6: 115,931,927-115,938,829. Applicants observed that micethat are homozygous for the targeted mutation are viable and fertile.Both the mutant and wildtype gene product (mRNA) is detected by cDNAsequencing chromatogram. The phenotype in heterozygous mice mimics theretinopathy and progressive retinal degeneration observed in patientswith autosomal dominant retinitis pigmentosa caused by the P23Hmutation. By 35 days of age, heterozygotes have a shorter rod outersegment when compared to controls. At post natal day 63, heterozygoteshave fewer rod nuclei (half the number observed in wildtype mice), anddecreased length of the rod outer segment. Homozygous mice exhibit amore severe phenotype with thinner outer nuclear layer, severephotoreceptor degeneration by post natal day 23 and loss of almost allphotoreceptor cells by post natal day 63. Glycosylation of the mutantP23H protein is severely diminished.

AAV Delivery of Cas9 System to the Retina Neurons in the Mouse StrainB6.129S6(Cg)-Rhotm 1.1Kpal/J:

Applicants chose to target the gene Rho that is critical for the normalfunction of retina neurons, and induce homologous recombination on thetarget to correct the related disease-relevant mutations P23H usingAdeno-associated virus (AAV) delivery of cas9 genome engineering toolsand a recombination template. Applicants designed three targets, P23Hmutation site labeled in orange with the single nucleotide mutation C-Alabeled in red. (see FIG. 18A). This approach demonstrates that thecorrection of the genetic mutation in this mouse model can rescue thedisease-associated phenotypes and further demonstrates the feasibilityof performing genome modification in neurons of adult animals. Further,the study provides information to assess the level of homologousrecombination that could be induced in neuronal cell types for geneticengineering purposes, using the retina neurons in this strain as a modelsystem. The experimental set up is as shown in the table below:

Injection Mice per Group purpose Routes Number of groups group TotalNeg. ctrl. - saline IV 1 group, 3 time 5 15 injection points Control -non-targeting IV 1 group, 3 time 5 15 vector points Targeting vector IV3 time points X 3 5 45 conditions

In total, 25 mice are used to set up breeder pairs so in total=100 micefor each strain are used for this study.

Retina Injection of AAV:

Subretinal injection is used as the delivery route. Mice receivesubretinal injection of 0.5-1 microliters of saline solution (0.9%sodium chloride) warmed to body temperature and containing up to 1E12viral particles. These mice are more than 6 weeks old. The animal iskept warm using a heating pad and/or lamp and is monitored. Animals aremonitored daily for clinical signs of procedure related complications.Up to two injection per mouse are administered.

Tissue Collection:

After 1 to 4 weeks after injections of materials, mice are sacrificed bythe CO₂ inhalation method prior to tissue sampling from mice. Tissuesare analyzed for evidence of successful gene transfection, genomealterations, or toxicity.

Example 3: In Vivo Therapeutic Genome Engineering Approach for RetinitisPigmentosa

Guide Selection and In Vitro Validation

First, guides for SaCas9 that target the gene RHO in human genome aredesigned as shown below. It has been selected based on the locus ofdisease-causing mutation P23H. The design was generated throughcomputational algorithm to maximize the potential of introducingon-target cleavage that is closest to the disease mutation site tofacilitate highest efficiency and specificity of therapeutic genecorrection. Guides were also screened for their DNaseI hyper-sensitivity(HS) assay results to maximize the accessibility of the genomic regioncorresponding to the target guide sequence, thereby increasing theexpected efficiency of the guides following delivery in vivo. More thanone guides are selected and then screened using Surveyor assay in vitroin cultured human cells (HEK 293FT) to measure their efficiency forinducing indel formation at target genomic locus. The guide with highestefficiency in vitro is then selected for virus production and downstreamexperiment in vivo. FIG. 18A-B shows the guide design for RHO locus, andin vitro guide screening results using the Surveyor assay.

Homologous Recombination Template Design and Validation

Secondly, to induce homologous recombination on the target to correctthe related disease-relevant mutations P23H using homology-directedrepair, a HR vector is synthesized based on the wild type (normal)genomic sequence of the unaffected human individual. This vector bearsthe AAV packaging signal, and the normal version of genomic sequencewith up to 5 kb total homology arms: two homology arms, left and right,are on each side of the sequence that sandwich the target mutation siteP23H in the middle, as shown below. This vector is validated in vitro byco-transfecting at 1:1, 1:3, 1:5 ratio with the corresponding vectorencoding the SaCas9 system with the best guide as measured in previoussection, then the homologous recombination (HR) efficiency at targetP23H locus is measured using restriction fragment length polymorphismassay (RFLP) assay and thus validate the optimal condition for the HDRprocedure.

FIG. 19 shows an RHO HR AAV vector. The specific sequence for the partresponsible for serving as homologous recombination template is listedbelow.

>Rho HR AAV Vector Template Region (SEQ ID NO: 124)ACCAGAAAGTCTCTAGCTGTCCAGAGGACATAGCACAGAGGCCCATGGTCCCTATTTCAAACCCAGGCCACCAGACTGAGCTGGGACCTTGGGACAGACAAGTCATGCAGAAGTTAGGGGACCTTCTCCTCCCTTTTCCTGGATCCTGAGTACCTCTCCTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCCCTCTTAGAAGCCAATTAGGCCCTCAGTTTCTGCAGCGGGGATTAATATGATTATGAACACCCCCAATCTCCCAGATGCTGATTCAGCCAGGAGCTTAGGAGGGGGAGGTCACTTTATAAGGGTCTGGGGGGGTCAGAACCCAGAGTCATCCAGCTGGAGCCCTGAGTGGCTGAGCTCAGGCCTTCGCAGCATTCTTGGGTGGGAGCAGCCACGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGACGGGTGTGGTACGCAGCCCCTTCGAGTACCCACAGTACTACCTGGCTGAGCCATGGCAGTTCTCCATGCTGGCCGCCTACATGTTTCTGCTGATCGTGCTGGGCTTCCCCATCAACTTCCTCACGCTCTACGTCACCGTCCAGCACAAGAAGCTGCGCACGCCTCTCAACTACATCCTGCTCAACCTAGCCGTGGCTGACCTCTTCATGGTCCTAGGTGGCTTCACCAGCACCCTCTACACCTCTCTGCATGGATACTTCGTCTTCGGGCCCACAGGATGCAATTTGGAGGGCTTCTTTGCCACCCTGGGCGGTATGAGCCGGGTGTGGGTGGGGTGTGCAGGAGCCCGGGAGCATGGAGGGGTCTGGGAGAGTCCCGGGCTTGGCGGTGGTGGCTGAGAGGCCTTCTCCCTTCTCCTGTCCTGTCAATGTTATCCAAAGCCCTCATATATTCAGTCAACAAACACCATTCATGGTGATAGCCGGGCTGCTGTTTGTGCAGGGCTGGCACTGAACACTGCCTTGATCTTATTTGGAGCAATATGCGCTTGTCTAATTTCACAGCAAGAAAACTGAGCTGAGGCTCAAAGAAGTCAAGCGCCCTGCTGGGGCGTCACACAGGGACGGGTGCAGAGTTGAGTTGGAAGCCCGCATCTATCTCGGGCCATGTTTGCAGCACCAAGCCTCTGTTTCCCTTGGAGCAGCTGTGCTGAGTCAGACCCAGGCTGGGCACTGAGGGAGAGCTGGGCAAGCCAGACCCCTCCTCTCTGGGGGCCCAAGCTCAGGGTGGGAAGTGGATTTTCCATTCTCCAGTCATTGGGTCTTCCCTGTGCTGGGCAATGGGCTCGGTCCCCTCTGGCATCCTCTGCCTCCCCTCTCAGCCCCTGTCCTCAGGTGCCCCTCCAGCCTCCCTGCCGCGTTCCAAGTCTCCTGGTGTTGAGAACCGCAAGCAGCCGCTCTGAAGCAGTTCCTTTTTGCTTTAGAATAATGTCTTGCATTTAACAGGAAAACAGATGGGGTGCTGCAGGGATAACAGATCCCACTTAACAGAGAGGAAAACTGAGGCAGGGAGAGGGGAAGAGACTCATTTAGGGATGTGGCCAGGCAGCAACAAGAGCCTAGGTCTCCTGGCTGTGATCCAGGAATATCTCTGCTGAGATGCAGGAGGAGACGCTAGAAGCAGCCATTGCAAAGCTGGGTGACGGGGAGAGCTTACCGCCAGCCACAAGCGTCTCTCTGCCAGCCTTGCCCTGTCTCCCCCATGTCCAGGCTGCTGCCTCGGTCCCATTCTCAGGGAATCTCTGGCCATTGTTGGGTGTTTGTTGCATTCAATAATCACAGATCACTCAGTTCTGGCCAGAAGGTGGGTGTGCCACTTACGGGTGGTTGTTCTCTGCAGGGTCAGTCCCAGTTTACAAATATTGTCCCTTTCACTGTTAGGAATGTCCCAGTTTGGTTGATTAACTATATGGCCACTCTCCCTATGGAACTTCATGGGGTGGTGAGCAGGACAGATGTCTGAATTCCATCATTTCCTTCTTCTTCCTCTGGGCAAAACATTGCACATTGCTTCATGGCTCCTAGGAGAGGCCCCCACATGTCCGGGTTATTTCATTTCCCGAGAAGGGAGAGGGAGGAAGGACTGCCAATTCTGGGTTTCCACCACCTCTGCATTCCTTCCCAACAAGGAACTCTGCCCCACATTAGGATGCATTCTTCTGCTAAACACACACACACACACACACACACACAACACACACACACACACACACACACACACACACACAAAACTCCCTACCGGGTTCCCAGTTCAATCCTGACCCCCTGATCTGATTCGTGTCCCTTATGGGCCCAGAGCGCTAAGCAAATAACTTCCCCCATTCCCTGGAATTTCTTTGCCCAGCTCTCCTCAGCGTGTGGTCCCTCTGCCCCTTCCCCCTCCTCCCAGCACCAAGCTCTCTCCTTCCCCAAGGCCTCCTCAAATCCCTCTCCCACTCCTGGTTGCCTTCCTAGCTACC CTC

AAV Delivery of Cas9 Genome Engineering Tools and a RecombinationTemplate

Finally, for employing the Adeno-associated virus (AAV) delivery of cas9genome engineering tools and a recombination template in vivo, bothviruses are produced with serotype AAV1, AAV2, AAV5, AAV7, AAV8 (all areeffective), purified by gradient ultracentrifugation or chromatographymethods. The virus particles are then injected using the optimalcondition as determined in previous section through the subretinal routefor delivery to the photoreceptor and RPE cells.

Detailed Injection Protocol

Retina injection of AAV: subretinal injection is performed by injectionof up to 1 milliliter of saline solution (0.9% sodium chloride)containing different dose of AAV viral particles warmed to bodytemperature. The two different AAV vector, one for SaCas9 and one for HRtemplate are mixed at different ratios (e.g. SaCas9 vs. HR template=1:1,1:3, or 1:5) to determine the optimal condition. The dose used for theinjection can be as low as 1.5×10E10 vector genomes total, or up to1.5×10E11 vector genomes total. The higher dose gives better genetherapy effectiveness but may lead to higher chance of complications dueto immune response to the viral vector injection. Patients are monitoreddaily for clinical signs of procedure related complications. Thisprocedure essentially follows guidelines provided in the followingpublications: Maguire A. M. et al. N Engl. J. Med. 2008 May 22;358(21):2240-8. doi: 10.1056/NEJMoa0802315. Epub 2008 Apr. 27. Safetyand efficacy of gene transfer for Leber's congenital amaurosis.Simonelli F. et al. Mol. Ther. 2010 March; 18(3):643-50. doi:10.1038/mt.2009.277. Epub 2009 Dec. 1. Gene therapy for Leber'scongenital amaurosis is safe and effective through 1.5 years aftervector administration. Maguire A. M. et al. Lancet. 2009 Nov. 7;374(9701):1597-605. doi: 10.1016/S0140-6736(09)61836-5. Epub 2009 Oct.23. Age-dependent effects of RPE65 gene therapy for Leber's congenitalamaurosis: a phase 1 dose-escalation trial.

Post Injection Procedures

Post injection, the patient is monitored for any immunological responseor adverse effect. After at least 4 weeks, restriction fragment lengthpolymorphism assay (RFLP) is used to assess the level of homologousrecombination that could be induced in ocular cell types. And the reliefor recovery of RP phenotype is then also evaluated.

Example 4: In Vivo Therapeutic Genome Engineering for Achromatopsia

Guide Selection and In Vitro Validation

First, guides for SaCas9 that target the gene CNGA3 and CNGB3 in humangenome are designed as shown below. It has been selected based on thelocus of disease-causing mutation, i.e. R277C and R283W for CNGA3 and1148delC for CNGB3. The design was generated through computationalalgorithm to maximize the potential of introducing on-target cleavagethat is closest to the disease mutation site to facilitate highestefficiency and specificity of therapeutic gene correction. Guides werealso screened for their DNaseI hyper-sensitivity (HS) assay results tomaximize the accessibility of the genomic region corresponding to thetarget guide sequence, thereby increasing the expected efficiency of theguides following delivery in vivo. More than one guides are selected sothat the most efficient guides can be tested in vitro via Surveyorassay. The guide with highest efficiency in vitro is then selected forvirus production and downstream experiment in vivo. FIG. 20A-B showsguide selection for CNGA3 and CNGB3.

Homologous Recombination Template Design and Validation

Second, to induce homologous recombination on the target to correct therelated disease-relevant mutations using homology-directed repair, a HRvector need to be synthesized based on the wild type (normal) genomicsequence of the CNGA3 and CNGB3 genes from unaffected human individual,respectively.

FIGS. 21 and 22 show maps of the CNGA3 HR AAV vector and CNGB3 HR AAVvector, respectively. These vectors bear the AAV packaging signal, andthe normal version of genomic sequence with up to 5 kb total homologyarms: two homology arms, left and right, are on each side of thesequence that sandwich the target mutation site in the middle, as shownbelow. This vector is validated in vitro by co-transfecting at 1:1, 1:3,1:5 ratio with the corresponding vector encoding the SaCas9 system withthe best guide as measured in previous section, then the homologousrecombination (HR) efficiency at target locus is measured usingrestriction fragment length polymorphism assay (RFLP) assay and thusvalidate the optimal condition for the HDR procedure.

The specific sequence for the part responsible for serving as homologousrecombination template is listed below.

>CNGA3 HR AAV Vector Template Region (SEQ ID NO: 125)CTGCTGCCTGCTCTGTCCCCTTTAAGTATTGACATCCTCAAAACCCTCTTTGGAAAAAGCACAGGCCACAGATCTTACTGTGACTTGTGTTTCTTTCTCCTAGGTGTACCTTCAACCTTGATAAAAATAAACCTCTAAATCAATTGAGATCTGCCTCCGTCACTTTTTTTTTTTCAAAGACTCAGAGTCTCACTCTGTTGCCCAGGCTGGAGTGTAGTGGTGCGATCTTGGCTCATTGCACCCTCCACCTCCTGGGTTCAAGTGGTTCTCGTGCCTCAGCTTCCTGAGTAGCTGGGATTACAGGGGTGCACCACCACATCTGGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGCCCAGGCTGGTCTCAAACCCTTGACCTCAGGTGATCCACCCGCCTCGGCCTCTCAAAGTACTGGGATTATAGGCATGAGCCACGGCACCCGGCCCTCTGTCACTTTTTGATTTACAACATGTATCTCTAATTTTAAAGGATCCTTTTTTAAAATATGTATATAATTTCCATTTATCTTTTAAAATTTAATAATCATTCTTTGTTATCATGTAATACCCAATTTATATTTAAATTTACTCAATCAACCTATGTTTTAAAAAAATTCAATAGAATAGATTAGAACCTCATAGAATAGAAAATATCAGAGTGCATTTCCTGTAGTAATGGTAAGTGTTGTTTTTGAAATCATTTCTATTATATATGTATCACTGCATACTGTGTAGCCGTGAGGTAAAATATGTTTCTTTGTACTATGGTCAAAAAAAGTCAGCCTCTGTGATGCCCAATGACCTCCATCTTCTTCTTTAGGTTTTCTCGAGCAAGGCTTAATGGTCAGTGATACCAACAGGCTGTGGCAGCATTACAAGACGACCACGCAGTTCAAGCTGGATGTGTTGTCCCTGGTCCCCACCGACCTGGCTTACTTAAAGGTGGGCACAAACTACCCAGAAGTGAGGTTCAACCGCCTACTGAAGTTTTCCCGGCTCTTTGAATTCTTTGACCGCACAGAGACAAGGACCAACTACCCCAATATGTTCAGGATTGGGAACTTGGTCTTGTACATTCTCATCATCATCCACTGGAATGCCTGCATCTACTTTGCCATTTCCAAGTTCATTGGTTTTGGGACAGACTCCTGGGTCTACCCAAACATCTCAATCCCAGAGCATGGGCGCCTCTCCAGGAAGTACATTTACAGTCTCTACTGGTCCACCTTGACCCTTACCACCATTGGTGAGACCCCACCCCCCGTGAAAGATGAGGAGTATCTCTTTGTGGTCGTAGACTTCTTGGTGGGTGTTCTGATTTTTGCCACCATTGTGGGCAATGTGGGCTCCATGATCTCGAATATGAATGCCTCACGGGCAGAGTTCCAGGCCAAGATTGATTCCATCAAGCAGTACATGCAGTTCCGCAAGGTCACCAAGGACTTGGAGACGCGGGTTATCCGGTGGTTTGACTACCTGTGGGCCAACAAGAAGACGGTGGATGAGAAGGAGGTGCTCAAGAGCCTCCCAGACAAGCTGAAGGCTGAGATCGCCATCAACGTGCACCTGGACACGCTGAAGAAGGTTCGCATCTTCCAGGACTGTGAGGCAGGGCTGCTGGTGGAGCTGGTGCTGAAGCTGCGACCCACTGTGTTCAGCCCTGGGGATTATATCTGCAAGAAGGGAGATATTGGGAAGGAGATGTACATCATCAACGAGGGCAAGCTGGCCGTGGTGGCTGATGATGGGGTCACCCAGTTCGTGGTCCTCAGCGATGGCAGCTACTTCGGGGAGATCAGCATTCTGAACATCAAGGGGAGCAAGTCGGGGAACCGCAGGACGGCCAACATCCGCAGCATTGGCTACTCAGACCTGTTCTGCCTCTCAAAGGACGATCTCATGGAGGCCCTCACCGAGTACCCCGAAGCCAAGAAGGCCCTGGAGGAGAAAGGACGGCAGATCCTGATGAAAGACAACCTGATCGATGAGGAGCTGGCCAGGGCGGGCGCGGACCCCAAGGACCTTGAGGAGAAAGTGGAGCAGCTGGGGTCCTCCCTGGACACCCTGCAGACCAGGTTTGCACGCCTCCTGGCTGAGTACAACGCCACCCAGATGAAGATGAAGCAGCGTCTCAGCCAACTGGAAAGCCAGGTGAAGGGTGGTGGGGACAAGCCCCTGGCTGATGGGGAAGTTCCCGGGGATGCTACAAAAACAGAGGACAAACAACAGTGAAAATGCAGCATCTGTCTCCTGCTTCACAGGGTCGACTGTCAGGGTGACCGTATGTGGCCGCAGCTGTGTGGCATGGAACTTGGTCAGGGTTGAATTCCAGCTCTACTCACCCTTTGAAAGCTGTGTGACTGCCTGAGAGAACCTGTTTCTTCACCTAAAAAATGGGACTTTTTGTCTCAGTCCCAGTGAAGTGCCAGGTTTGATTGTGAAGTCCGCATGAAACACTGCACCAGGCAGGGCTTTGCAAAGTGCAA >CNGB3 HR AAV Vector Template Region(SEQ ID NO: 126) ACTTTGAGGCAATTTTACTGTAGCTGGTATTTTAGTCAATTTTTAGATAAATTAGTTGTTTATATCAAAGTAAATAATTCACATTCTAAAGGGAATTATTTATTTAGTAAATTTTCTGGAAATTGAGTGTCTGTGTGTGTGTGTTTCCCAATCAGTGGTCCTTCTGACTTTAAATTCTTTAAAATCGGTTCTGGTTGTTATAATCCCTTATACATATCCAACTCACTCTAGGTAGTATGTAATTTTGTAAGTTATTTTCCCTCTCTTTGCTCTATCCTATAATTGCTCTCCATCCCAAGGCTGCAGTGAGTTGCCCTTCAAAGTAATGCTGGGACCTGCTTTTTTTCCAGTTTGGACATTGCCTTATTATATGTTCAATGTCATTTCACTGGAGCAGAAAGTTAGTGAAGTCAACTTTATGCCAGGTCTTTGTATTTTACCAAAAGGAAATTTCACTATTAAATAACCCAGTTGCCATTTCTGAGTCCTGATTCTACTGTTCTAAATTTTTCAAGTGATCTTTTTTTATTTCTGGGACACTTGCATACCTAATTGTCAAGTTTAATTTATGATCCTCGTTACTCTCTAAGTGTTTAATTGAGTTAGTGGTTATAGCTGACTCATAAACCCATAAAACCCTTCACTGGTAAACTAATTAGCCACTGCACCTGCCCTTTAAATAATTAACATTGTTCATTACTAACAATCGGCATCGGAGTTATTAAAAGTTACCTTACTGCTCAGTTGTCTAGAGGCTTTCAAACTTTTTTGATCATGATCCAGAGTAAAAAATGCATTTCACAGGCCAACTCAGGATACACACACACACACACACACTCCCCTACTATCTATCAGACTCTGATATTTTCTATTCTATTATTCTCTATCTTCTTTCATTTAAAAATGTATTGACTTACTAAAGAGGTTTTTCAGCTTAAAAATTTTTATTTAGACCAATTCATGGGGTACTCATGCAATTTTGTTACATGTATACAATGCATAGTGATCAAGTCAGGGTGTTTAGGGTGTTTATTACTCGAGTACAATACATGTTTTGAAACTATAGTCACCCTACTCTGTTGCAAACGTTGAATATATTCTTACTGTATGTTTGTATCCTTTAATCCACTTTTCTTTATGCGCTCCTCTCCCCACCACTCACCCTTCCCAGTCTCTGTTATCTTTCCACTCTCTGTCTCTATGTGATCAAAATTTTTAGCTCCCACATATATGTGAGAACGTGTGATATTTATCGTTTTGGGTCTGGCTTATTTCACTAAGATAATGACCTTCAGTCCCATCCATTTTGGTGCAAATGACATGATTTTATCCTTTTTTATGGCCAAATAGCATTTACCAGCCATTGAATGGGTTATGACAGCTTCAAAAACACTGGCTCTCATAAATTCATACAATGAAACAGAATGTTAAAAATAATCAATAAAGGTTTCTTTCAAAATCAGAACTTACTCGTTTCCTTCCCCATCATACACCCATCTAGTAGTGCCAATTCCTTCATAGTTTGAAGCCCAGTAATAAACACAGGCATTAATGTGCAGAATAAACAGCAAGTATCCAGTTGTTCGAATAACTCTGTCAGAGAGAATAGATGCAAAGTAAGATTCATGTTGTTTCTGAAATACAGCCTATTTTAACATTTTCTTTTCCTTAAAGTCACCAGCGAACCCCTTGCTTTGGATTTGTGAACTGTTAACTCTCATTAGTACAGTACAAAGTGATGGTGCCATTGCATGTTTTCTGATGGCAATGTCTTGACTGGGATTGACAGAGTGTAAAGAAAAAAAAAAGAAAAAGAAAACTTCCTCTCTTTTCACAGATGTTGTGAGTCAACTCCGTGAAAGACATGCCTCAAAGGTCACTTCTTCAGTTTAAGTCCCATAAAATACACTATGCTAATTTAACTGGATATCTCTGAAAAGCTCATGAGACTTTATGCTACGATGAATGGCAACTAGAGGTTTCGGTGCAAGTAAAATTTAGAACAACAAACGAATGAAATTCAGATTAGGAATGAATTATCATGAGAAAGGTTTAAAGTTAACTTGCAAAAGAGTATGTTTTTCTGTACTTGTTTTGACAGAGGCAGATAATAAGTCCTATTTTCTTAGTCCAGTATTCTAAAATCTGATATGATTTTCATACTCTTATTTCACTTAAAATATCCACATCTGTTCTAGAACATAGTCCTATATTTTATATAGCCAAAGCTGAAATTATATCCTTTTTTTGAAGAGGGGGGTCATATCCCTGCCAAATTCCGTCTAAAATGTTGTACCATTGCTTTTTCCCCTTCCCCCAAGTATACTGAGTTATACTTTACCTGTAGATATATGCTTTGTCCATTATAGACTCTAGGTGATGATTAAATTCAAAAAATGAAGTGTACTATATAGAAAAGCAAAAGAAATCCAAAAGCATGTTAGTCTTAAATATATATATTTAAATAAAACTATATGAAATAGATTTTATTACTGAAAAT

AAV Delivery of Cas9 Genome Engineering Tools and a RecombinationTemplate

Finally for employing the Adeno-associated virus (AAV) delivery of cas9genome engineering tools and a recombination template in vivo, bothviruses are produced with serotype AAV1, AAV2, AAV5, AAV7, AAV8 (all areeffective), purified by gradient ultracentrifugation or chromatographymethods. The virus particles are then injected using the optimalcondition as determined in previous section through the subretinal routefor delivery to the photoreceptor and RPE cells.

Detailed Injection Protocol

Retina injection of AAV: subretinal injection is performed by injectionof up to 1 milliliter of saline solution (0.9% sodium chloride)containing different dose of AAV viral particles warmed to bodytemperature. The two different AAV vector, one for SaCas9 and one for HRtemplate are mixed at different ratios (e.g. SaCas9 vs. HR template=1:1,1:3, or 1:5) to determine the optimal condition. The dose used for theinjection can be as low as 1.5×10E10 vector genomes total, or up to1.5×10E11 vector genomes total. The higher dose may give better genetherapy effectiveness but might lead to higher chance of complicationsdue to immune response to the viral vector injection. Patients aremonitored daily for clinical signs of procedure related complications.

Post Injection Procedures

Post injection, the patient is monitored for any immunological responseor adverse effect. After at least 4 weeks, restriction fragment lengthpolymorphism assay (RFLP) is be used to assess the level of homologousrecombination that could be induced in ocular cell types. And the reliefor recovery of Achromatopsia phenotype is then also evaluated.

Example 5: In Vivo Therapeutic Genome Engineering Approach forAge-Related Macular

Degeneration

Guide Selection and In Vitro Validation

First, guides for SaCas9 that target the genomic locus VEGFA in humangenome are designed as shown below. It has been selected within thefirst exon of the gene, but other parts of the gene can also be targetedas well so that a wide range of target region can be screened todetermine the most effective guide design. All the designs weregenerated through computational algorithm to maximize the potential ofintroducing on-target cleavage that is closest to the transcriptionalstart site and located within the common region of different expressedtranscripts in retina. This will facilitate highest efficiency andspecificity of therapeutic gene correction. Guides were also screenedfor their DNaseI hyper-sensitivity (HS) assay results to maximize theaccessibility of the genomic region corresponding to the target guidesequence, thereby increasing the expected efficiency of the guidesfollowing delivery in vivo. More than one guides are selected so thatthe most efficient guides can be tested in vitro. FIG. 23A-B shows guideselection for VEGFA.

To validate that the designed guides can effective repress theexpression of VEGFA gene in human cells and finding the optimal guidesand conditions. The closed AAV vector bearing the designed guides aredelivered in vitro into human cells (e.g. HEK293). The cells wereharvested 72-96 hours post delivery. RNAs and proteins are extractedfrom the cells. The mRNA level of VEGF is measured via qRT-PCR method orother RNA measurement method, whereas protein level of VEGF is alsomeasured via ELISA or other protein measurement method. The mostcritical criterion is the VEGF protein level because this is directlyrelevant for the clinical effectiveness of the system. The guide withhighest efficiency in vitro to lower the expression level of VEGF isthen selected for virus production and downstream experiment in vivo.

Finally for employing the Adeno-associated virus (AAV) delivery of cas9genome engineering tools to disrupt VEGF expression in vivo, viruses areproduced with serotype AAV2 and AAV8 (both are effective, other AAVserotype such as AAV1, AAV5, AAV7, AAV9, or AAV-DJ might be used butwill be less potent). The viral particles are all purified by gradientultracentrifugation or chromatography methods.

The AAV virus particles are then injected using the optimal condition asdetermined in previous section through the intravitreal route, where AAVis injected in the vitreous humor of the eye. This procedure is lessinvasive and can sustain persistent expression of the construct toobtain sufficient disruption of VEGF expression to induce therapeuticcorrective effects.

Detailed Injection Protocol

Retina injection of AAV: intravitreal injection is performed byinjection of typically 100 microliter (or up to 1 milliliter but notrecommended) of saline solution (0.9% sodium chloride) containingdifferent dose of AAV viral particles warmed to body temperature. Thedose used for the injection can be as low as 1×10E8 vector genomes intotal, or up to 1×10E11 vector genomes in total. The use of differentdosage allows the determination of optimal dose.

The higher dose will give better gene therapy effectiveness but mightlead to higher chance of complications due to immune response to theviral vector injection. Patients will be monitored daily for clinicalsigns of procedure related complications.

Reference for human injection parameter is available at Clinical Trials(dot) Gov—the US Government website for information on clinical trials,having a web address of: clinicaltrials.gov/ct2/show/NCT01024998.

Post Injection Procedures

Post injection, the patient are monitored for any immunological responseor adverse effect. After at least 4 weeks, ELISA or other proteinmeasurement methods can be employed to assess the level of VEGF in theretina. And the relief or recovery of ARMD phenotype is then evaluated.

Key safety measures to be followed in the patients include the maximumtolerated dose of a single uniocular intravitreal injection, number oftreatment emergent adverse events, and the thickness of the retina postinjection.

Example 6: dSaCas9 to Stimulate ATOH1 Expression to Treat Deafness orHearing Impairments

Guide Selection and In Vitro Validation

First, guides for SaCas9 that target the gene ATOH1 in human genome aredesigned as shown below. It has been selected based on the optimalparameters to maximize the efficiency of epigenetic modulation on targetgene expression. Specifically, the design was generated throughcomputational algorithm to maximize the potential of introducingon-target binding to facilitate highest efficiency and specificity oftherapeutic gene therapy. Guides were also screened for their DNaseIhyper-sensitivity (HS) assay results to maximize the accessibility ofthe genomic region corresponding to the target guide sequence, therebyincreasing the expected efficiency of the guides following delivery invivo. Moreover, transcription factor sites that potentially mightinterfere with the binding were also considered during the selection ofthe guides. More than one guides are selected and then screened in vitroin cultured human cells (HEK 293FT) to measure their efficiency forinducing ATOH1 gene expression. The guide with highest efficiency invitro is then selected for downstream experiment and therapeuticintervention in vivo. The specific design of guides for ATOH1 is shownat FIG. 25A-C.

Guide Screening and Validation

To validate that the designed guides can effective induce the expressionof ATOH1 gene in human cells and finding the optimal guides andconditions, in vitro screening and verification is performed with humancell lines. The system, which contains dSaCas9, fusion effector, andoptimal guide RNAs, is delivered in vitro into human cells (e.g. HEK293)(FIG. 24). The cells were harvested 72-96 hours post delivery. RNAs andproteins are extracted from the cells. The mRNA level of ATOH1 ismeasured via qRT-PCR method or other RNA measurement methods, whereasprotein level of ATOH1 is measured via ELISA or other proteinmeasurement methods. The most critical criteria is the ATOH1 proteinlevel because this is directly relevant for the clinical effectivenessof the system. The guide with highest efficiency in vitro to stimulatethe expression of ATOH1 is then selected for in vivo delivery andtherapeutic demonstration.

AAV or Ad viral particles or other delivery vehicle packaging the entiresystem are then produced, and injected using the optimal conditions asdetermined experimentally. The most critical parameter to optimize isthe amount (i.e. dosage) of the delivery vehicle used for eachindividual injection.

The higher dose will give better gene therapy effectiveness but mightlead to higher chance of complications due to immune response to thevehicle injection or the off-target effects of the system. Patients aremonitored daily for clinical signs of procedure related complications.

Post Injection Procedures

Post injection, the patient are monitored for any immunological responseor adverse effect. Each week post injection, ELISA or other proteinmeasurement methods can be employed to assess the level of ATOH1stimulation. New hair cell growth in the Cochleae can be visualizedthrough biopsy and imaging methods. And the relief or recovery ofdeafness and hearing impairments is then evaluated through hearing testson the human patients.

Key safety measures to be followed in the patients include the maximumtolerated dose of a single injection into the human Cochleae, number oftreatment emergent adverse events post injection.

The invention is further described by the following numbered paragraphs:

1. A method of modeling a disease associated with a genomic locus in aeukaryotic organism or a non-human organism comprising manipulation of atarget sequence within a coding, non-coding or regulatory element ofsaid genomic locus comprising delivering a non-naturally occurring orengineered composition comprising:

-   -   (A)—I. a CRISPR-Cas system RNA polynucleotide sequence, wherein        the polynucleotide sequence comprises:        -   (a) a guide sequence capable of hybridizing to the target            sequence,        -   (b) a tracr mate sequence, and        -   (c) a tracr sequence, and    -   II. a polynucleotide sequence encoding Cas9, optionally        comprising at least one or more nuclear localization sequences,        wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and wherein the CRISPR complex comprises Cas9        complexed with (1) the guide sequence that is hybridized to the        target sequence, and (2) the tracr mate sequence that is        hybridized to the tracr sequence and the polynucleotide sequence        encoding Cas9 is DNA or RNA,        or    -   (B) I. polynucleotides comprising:        -   (a) a guide sequence capable of hybridizing to the target            sequence, and        -   (b) at least one or more tracr mate sequences,        -   II. a polynucleotide sequence encoding Cas9, and        -   III. a polynucleotide sequence comprising a tracr sequence,            wherein when transcribed, the tracr mate sequence hybridizes            to the tracr sequence and the guide sequence directs            sequence-specific binding of a CRISPR complex to the target            sequence, and            wherein the CRISPR complex comprises the Cas9 complexed            with (1) the guide sequence that is hybridized to the target            sequence, and (2) the tracr mate sequence that is hybridized            to the tracr sequence, and the polynucleotide sequence            encoding Cas9 is DNA or RNA.

2. The method of paragraph 1, wherein the Cas9 is SaCas9.

3. The method of paragraph 1 or 2, wherein the polynucleotides encodingthe sequence encoding Cas9, the guide sequence, tracr mate sequence ortracr sequence is/are RNA and are delivered via liposomes,nanoparticles, cell penetrating peptides, exosomes, microvesicles, or agene-gun.

4. The method of any of paragraphs 1 to 3, wherein the polynucleotidesare comprised within a vector system comprising one or more vectors.

5. A method of modeling a disease associated with a genomic locus in aeukaryotic organism or a non-human organism comprising manipulation of atarget sequence within a coding, non-coding or regulatory element ofsaid genomic locus comprising delivering a non-naturally occurring orengineered composition comprising a viral vector system comprising oneor more viral vectors operably encoding a composition for expressionthereof, wherein the composition comprises:

-   -   (A) a non-naturally occurring or engineered composition        comprising a vector system comprising one or more vectors        comprising    -   I. a first regulatory element operably linked to a CRISPR-Cas        system RNA polynucleotide sequence, wherein the polynucleotide        sequence comprises        -   (a) a guide sequence capable of hybridizing to the target            sequence,        -   (b) a tracr mate sequence, and        -   (c) a tracr sequence, and    -   II. a second regulatory element operably linked to an        enzyme-coding sequence encoding Cas9, optionally comprising at        least one or more nuclear localization sequences,        wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein components I and II are located on the same or different        vectors of the system,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the Cas9 complexed with (1)        the guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence,        or    -   (B) a non-naturally occurring or engineered composition        comprising a vector system comprising one or more vectors        comprising    -   I. a first regulatory element operably linked to        -   (a) a guide sequence capable of hybridizing to the target            sequence, and        -   (b) at least one or more tracr mate sequences,    -   II. a second regulatory element operably linked to an        enzyme-coding sequence encoding Cas9, and    -   III. a third regulatory element operably linked to a tracr        sequence, wherein components I, II and III are located on the        same or different vectors of the system, wherein when        transcribed, the tracr mate sequence hybridizes to the tracr        sequence and the guide sequence directs sequence-specific        binding of a CRISPR complex to the target sequence, and        wherein the CRISPR complex comprises the Cas9 complexed with (1)        the guide sequence that is hybridized to the target        sequence; (2) the tracr mate sequence that is hybridized to the        tracr sequence; and wherein the Cas9 is preferably SaCas9.

6. The method of paragraph 5, wherein one or more of the viral vectorsare delivered via liposomes, nanoparticles, exosomes, microvesicles, ora gene-gun.

7. A method of treating or inhibiting a condition or a disease caused byone or more mutations in a genomic locus in a eukaryotic organism or anon-human organism comprising manipulation of a target sequence within acoding, non-coding or regulatory element of said genomic locus in atarget sequence in a subject or a non-human subject in need thereofcomprising modifying the subject or a non-human subject by manipulationof the target sequence and wherein the condition or disease issusceptible to treatment or inhibition by manipulation of the targetsequence comprising providing treatment comprising:

delivering a non-naturally occurring or engineered compositioncomprising an AAV or lentivirus vector system, comprising one or moreAAV or lentivirus vectors operably encoding a composition for expressionthereof, wherein the target sequence is manipulated by the compositionwhen expressed, wherein the composition comprises:

-   -   (A) a non-naturally occurring or engineered composition        comprising a vector system comprising one or more vectors        comprising    -   I. a first regulatory element operably linked to a CRISPR-Cas        system RNA polynucleotide sequence, wherein the polynucleotide        sequence comprises        -   (a) a guide sequence capable of hybridizing to the target            sequence in a eukaryotic cell,        -   (b) a tracr mate sequence, and        -   (c) a tracr sequence, and    -   II. a second regulatory element operably linked to an        enzyme-coding sequence encoding Cas9 comprising at least one or        more nuclear localization sequences,        wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein components I and II are located on the same or different        vectors of the system,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the Cas9 complexed with (1)        the guide sequence that is hybridized to the arget sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence, or    -   (B) a non-naturally occurring or engineered composition        comprising a vector system comprising one or more vectors        comprising    -   I. a first regulatory element operably linked to        -   (a) a guide sequence capable of hybridizing to an target            sequence in a eukaryotic cell, and        -   (b) at least one or more tracr mate sequences,    -   II. a second regulatory element operably linked to an        enzyme-coding sequence encoding Cas9, and    -   III. a third regulatory element operably linked to a tracr        sequence,        wherein components I, II and III are located on the same or        different vectors of the system,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence,        wherein the CRISPR complex comprises Cas9 complexed with (1) the        guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence; and wherein the Cas9 is preferably SaCas9.

8. The method of any preceding claim, wherein the method is carried outin vitro, ex vivo or in vivo.

9. The method of any preceding claim including inducing expression.

10. The method of paragraph 1 wherein the condition or disease is anocular disease.

11. The method of paragraph 10 where the ocular disease is retinitispigmentosa or achromatopsia.

12. The method of any of paragraphs 4 to 8 wherein the viral vector isan AAV or lentiviral vector.

13. A method of delivering the Cas9 of any preceding claim, comprisingdelivering to a cell mRNA encoding the Cas9.

14. The method of any one of paragraphs 1 to 13, wherein thepolynucleotide or sequence encoding the Cas9 is delivered to the cell bydelivering mRNA encoding the Cas9 to the cell.

15. A method of preparing the AAV or lentivirus vector of paragraph 7comprising transfecting plasmid(s) containing or consisting essentiallyof nucleic acid molecule(s) coding for the AAV or lentivirus intoAAV-infected or lentivirus-infected cells, and supplying AAV AAV orlentivirus rep and/or cap and/or helper nucleic acid moleculesobligatory for replication and packaging of the AAV or lentivirus.

16. A method of preparing an AAV or lentivirus vector for use in themethod of claim 7, comprising transfecting plasmid(s) containing orconsisting essentially of nucleic acid molecule(s) coding for the AAV orlentivirus into AAV-infected or lentivirus-infected cells, and supplyingAAV AAV or lentivirus rep and/or cap and/or helper nucleic acidmolecules obligatory for replication and packaging of the AAV orlentivirus.

17. The method of paragraph 15 or 16 wherein the AAV or lentivirus repand/or cap obligatory for replication and packaging of the AAV orlentivirus are supplied by transfecting the cells with helper plasmid(s)or helper virus(es).

18. The method of paragraph 17 wherein the helper virus is a poxvirus,adenovirus, lentivirus, herpesvirus or baculovirus.

19. The method of paragraph 18 wherein the poxvirus is a vaccinia virus.

20. The method of any of paragraph 15 to 20 wherein the cells aremammalian cells.

21. The method of any of paragraphs 15 to 20 wherein the cells areinsect cells and the helper virus (where present) is baculovirus.

22. The method of any preceeding claim wherein the Cas9 is a wild type,truncated or a chimeric Cas9.

23. A composition as defined in any of paragraphs 1-22 for use inmedicine or in therapy.

24. A composition as defined in any of paragraphs 1-22 for use in amethod of modeling a disease associated with a genetic locus in aeukaryotic organism or a non-human organism comprising manipulation of atarget sequence within a coding, non-coding or regulatory element ofsaid genetic locus.

25. Use of a composition as defined in any of paragraphs 1-24 in ex vivoor in vivo gene or genome editing.

26. Use of a composition as defined in any of paragraphs 1-24 in themanufacture of a medicament for in vitro, ex vivo or in vivo gene orgenome editing or for use in a method of modifying an organism or anon-human organism by manipulation of a target sequence in a genomiclocus associated with a disease or in a method of treating or inhibitinga condition or disease caused by one or more mutations in a genomiclocus in a eukaryotic organism or a non-human organism.

27. A composition comprising:

-   -   (A)—I. a CRISPR-Cas system RNA polynucleotide sequence, wherein        the polynucleotide sequence comprises:        -   (a) a guide sequence capable of hybridizing to a target            sequence in a eukaryotic cell,        -   (b) a tracr mate sequence, and        -   (c) a tracr sequence, and    -   II. a polynucleotide sequence encoding Cas9, optionally        comprising at least one or more nuclear localization sequences,        wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises the Cas9 complexed with (1)        the guide sequence that is hybridized to the target sequence,        and (2) the tracr mate sequence that is hybridized to the tracr        sequence and the polynucleotide sequence encoding SaCas9 is DNA        or RNA,        or    -   (B) I. polynucleotides comprising:        -   (a) a guide sequence capable of hybridizing to an target            sequence in a eukaryotic cell, and        -   (b) at least one or more tracr mate sequences,    -   II. a polynucleotide sequence encoding Cas9, and    -   III. a polynucleotide sequence comprising a tracr sequence,        wherein when transcribed, the tracr mate sequence hybridizes to        the tracr sequence and the guide sequence directs        sequence-specific binding of a CRISPR complex to the target        sequence, and        wherein the CRISPR complex comprises Cas9 complexed with (1) the        guide sequence that is hybridized to the target sequence, (2)        the tracr mate sequence that is hybridized to the tracr        sequence, and the polynucleotide sequence encoding Cas9 is DNA        or RNA; and the Cas9 is preferably SaCas9.        for use in medicine or therapy; or for use in a method of        modifying an organism or a non-human organism by manipulation of        a target sequence in a genomic locus associated with a disease        or disorder; or for use in a method of treating or inhibiting a        condition caused by one or more mutations in a genetic locus        associated with a disease in a eukaryotic organism or a        non-human organism; or for use in in vitro, ex vivo or in vivo        gene or genome editing.

28. The composition of paragraph 27, wherein the polynucleotides arecomprised within a vector system comprising one or more vectors.

29. The method, use or composition of any of the preceding claims,wherein the CRISPR-Cas system RNA is a chimeric RNA (chiRNA).

30. The method, use or composition of any of the preceding claims,wherein the CRISPR-Cas system is a multiplexed SaCas9 enzyme systemfurther comprising multiple chimeras and/or multiple multiguidesequences and a single tracr sequence.

31. The method, use or composition according any of the precedingclaims, wherein the Cas9 is a nuclease directing cleavage of bothstrands at the location of the target sequence.

32. The method, use or composition according to any of the precedingclaims, wherein the Cas9 comprises one or more mutations.

33. The method, use or composition according to paragraph 32, whereinthe Cas9 comprises one or more mutations D10A, E762A, H840A, N854A,N863A or D986A.

34. The method, use or composition according to paragraph 32 wherein theone or more mutations is in a RuvC1 domain of the Cas9.

35. The method, use or composition according to paragraph 30, whereinthe Cas9 is a nickase directing cleavage at the location of the targetsequence.

36. The method, use or composition according to paragraph 35, whereinthe nickase is a double nickase.

37. The method, use or composition according to any preceding claimfurther comprising at least two or more NLS.

38. The method, use or composition according to any preceding claim,wherein the SaCas9 has one or more mutations in a catalytic domain,wherein when transcribed, the tracr mate sequence hybridizes to thetracr sequence and the guide sequence directs sequence-specific bindingof a CRISPR complex to the target sequence, and wherein the enzymefurther comprises a functional domain.

39. The method, use or composition according to paragraph 38, whereinthe functional domain is a transcriptional activation domain.

40. The method, use or composition according to paragraph 39, whereinthe transcriptional activation domain is VP64.

41. A therapeutic genome editing method for treating or inhibiting acondition or a disease caused by one or more mutations in a genomiclocus in a eukaryotic organism or a non-human organism comprisingmanipulation of a target sequence within a coding, non-coding orregulatory element of said genomic locus in a target sequence in asubject or a non-human subject in need thereof comprising modifying thesubject or a non-human subject by manipulation of the target sequenceand wherein the condition or disease is susceptible to treatment orinhibition by manipulation of the target sequence comprising providingtreatment comprising:

delivering a non-naturally occurring or engineered compositioncomprising an AAV or lentivirus vector system, comprising one or moreAAV or lentivirus vectors operably encoding a composition for expressionthereof, wherein the target sequence is manipulated by the compositionwhen expressed, wherein the composition comprises:

(A) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising

I. a first regulatory element operably linked to a CRISPR-Cas system RNApolynucleotide sequence, wherein the polynucleotide sequence comprises

(a) a guide sequence capable of hybridizing to the target sequence in aeukaryotic cell,

(b) a tracr mate sequence, and

(c) a tracr sequence, and

II. a second regulatory element operably linked to an enzyme-codingsequence encoding Cas9 comprising at least one or more nuclearlocalization sequences,

wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,

wherein components I and II are located on the same or different vectorsof the system,

wherein when transcribed, the tracr mate sequence hybridizes to thetracr sequence and the guide sequence directs sequence-specific bindingof a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the Cas9 complexed with (1) theguide sequence that is hybridized to the target sequence, and (2) thetracr mate sequence that is hybridized to the tracr sequence,

or

(B) a non-naturally occurring or engineered composition comprising avector system comprising one or more vectors comprising

I. a first regulatory element operably linked to

(a) a guide sequence capable of hybridizing to an target sequence in aeukaryotic cell, and

(b) at least one or more tracr mate sequences,

II. a second regulatory element operably linked to an enzyme-codingsequence encoding Cas9, and

III. a third regulatory element operably linked to a tracr sequence,

wherein components I, II and III are located on the same or differentvectors of the system,

wherein when transcribed, the tracr mate sequence hybridizes to thetracr sequence and the guide sequence directs sequence-specific bindingof a CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises Cas9 complexed with (1) the guidesequence that is hybridized to the target sequence, (2) the tracr matesequence that is hybridized to the tracr; and the Cas9 is preferablySaCas9.

42. The method of paragraph 41, wherein the condition or disease isretinitis pigentosa or achromatopsia.

43. The method of paragraph 41, wherein the AAV is AAV1, AAV2, AAV5,AAV7, AAV8, AAV DJ or any combination thereof.

44. A method of individualized or personalized treatment of a geneticdisease in a subject in need of such treatment comprising:

(a) introducing multiple mutations ex vito in a tissue, organ or a cellline comprising SaCas9-expressing eukaryotic cell(s), or in vivo in atransgenic non-human mammal having cells that express SaCas9, comprisingdelivering to cell(s) of the tissue, organ, cell or mammal the vector asherein-discussed, wherein the specific mutations or precise sequencesubstitutions are or have been correlated to the genetic disease;

(b) testing treatment(s) for the genetic disease on the cells to whichthe vector has been delivered that have the specific mutations orprecise sequence substitutions correlated to the genetic disease; and

(c) treating the subject based on results from the testing oftreatment(s) of step (b).

45. The method of paragraph 44, wherein the genetic disease is an oculardisease.

46. The method of paragraph 45, wherein the ocular disease is retinitispigmentosa or achromatopsia.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

What is claimed is:
 1. A therapeutic genome editing method for treatingor inhibiting an ocular disease in a subject, the method comprisinglocally delivering a pharmaceutical composition comprising AAV vectorsto the subject's eye via intravitreal injection or subretinal injection,wherein each of the AAV vectors encodes a CRISPR-Cas system thatcomprises: (A) one or more CRISPR-Cas system chimeric guide RNAs eachcomprising: a guide sequence capable of hybridizing to a target sequencein a genomic locus associated with the ocular disease, a tracr matesequence, and a tracr sequence, and (B) a Cas9 protein, wherein the Cas9protein is Staphylococcus aureus Cas9 (SaCas9) and comprises one or morenuclear localization sequences (NLSs), wherein each of the CRISPR-Cassystem chimeric guide RNAs forms a CRISPR complex with the SaCas9 anddirects sequence-specific binding of the CRISPR complex to the targetsequence adjacent to a SaCas9 protospacer adjacent motif (PAM)comprising NNGRR, whereby the SaCas9 cleaves the genomic locus, andwherein the composition is administered at a dose of about 1×10⁵ to1×10⁵⁰ viral genomes.
 2. The method of claim 1, wherein the CRISPR-Cassystem is a multiplexed CRISPR-Cas system comprising multiple guidesequences each capable of hybridizing to a different target sequence anda single tracr sequence.
 3. The method of claim 1, wherein the Cas9 is anuclease directing cleavage of both strands at the location of thetarget sequence.
 4. The method of claim 1, wherein the Cas9 comprisesone or more mutations, selected from D10A, E762A, H840A, N854A, N863A orD986A, or wherein the one or more mutations is in a RuvC1 domain of theCas9.
 5. The method of claim 1, wherein the Cas9 is a nickase directingcleavage at the location of the target sequence.
 6. The method of claim5, wherein the nickase is a double nickase.
 7. The method of claim 1,wherein the Cas9 protein comprises two or more NLSs.
 8. The method ofclaim 1, wherein the Cas9 has one or more mutations in a catalyticdomain, and wherein the Cas9 protein further comprises a functionaldomain.
 9. The method of claim 8, wherein the functional domain is atranscriptional activation domain.
 10. The method of claim 9, whereinthe transcriptional activation domain is VP64.
 11. The method of claim1, wherein the AAV vectors comprise AAV1, AAV2, AAV5, or any combinationthereof.
 12. The method of claim 1, wherein the ocular disease is aretinal disease.
 13. The method of claim 12, wherein the retinal diseaseis a hereditary retinal disease.
 14. The method of claim 1, wherein theCRISPR-Cas system further comprises a recombination template.
 15. Themethod of claim 1, wherein the ocular disease is Leber CongenitalAmaurosis (LCA), Retinitis Pigmentosa, age related macular degeneration,or Usher Syndrome.
 16. The method of claim 1, wherein about 1×10⁵ to1×10⁵⁰ viral genomes is delivered to the subject in a single dose.
 17. Atherapeutic genome editing method for treating a retinal disease in asubject, the method comprising locally delivering a pharmaceuticalcomposition comprising AAV vectors to the subject's eye via subretinalinjection, wherein each of the AAV vectors encodes a CRISPR-Cas systemthat comprises: (A) two or more CRISPR-Cas system chimeric guide RNAseach comprising: a guide sequence capable of hybridizing to a targetsequence in a genomic locus associated with the retinal disease, a tracrmate sequence, and a tracr sequence, and (B) a Cas9 protein, wherein theCas9 protein is Staphylococcus aureus Cas9 (SaCas9) and comprises one ormore nuclear localization sequences (NLSs); wherein each of theCRISPR-Cas system chimeric guide RNAs forms a CRISPR complex with theSaCas9 and directs sequence-specific binding of the CRISPR complex tothe target sequence adjacent to a SaCas9 protospacer adjacent motif(PAM) comprising NNGRR, whereby the SaCas9 cleaves the genomic locus,and wherein the AAV vectors are AAV1, AAV2, AAV5, or a combinationthereof.